haploid a- and -cells form shmoos in response to chemical signals shmoos mate to form diploid a/ ...
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Haploid a- and -cells form shmoos in response to chemical signals
Shmoos mate to form diploid a/ cell
Examples of:- “differentiated” cell types (a-, -, and a/-cells) cell-cell adhesion-cell-cell signaling
Human body consists of trillions of cells, 200+ specialized cell types that must differentiate (next time) and communicate (today) with one another
Cell-cell communication required to coordinate:- physiology and metabolism- behavior-growth, proliferation, and differentiation
ECB 16-1
Mating dance of a budding yeast (S.cerevisiae)…
Multicellularity: From cells to tissues to organisms
shmoos
“Neuronal”
Cell body of neuron
Post-synaptictarget (muscle,
neuron, etc)
Axon
Synapse
Action potential
Basic categories of cell-cell signaling in animals
ECB 16-3
“Paracrine” (local)ex. inflammation
Signaling cell
Target cells
“Autocrine”
“Contact-mediated” (short range)ex. - nerve cell production
Signaling cell
Target cell
(ex.-hormones)“Endocrine” (long distance)
Endocrine (signaling) cell
Target cells
Bloodstream
hormone
Cellular response depends on specific combination of signals
ECB 16-6
No signal often results in activation of apoptosis
Common features of cell-cell signaling pathways
Other signals
ECB 16-7
Receptors for diffusible signals can be intracellular or surface
Small non-polar molecules Large polar molecules
Plasma membran
e
cross plasma membrane by simple diffusion
And bind to intra- cellular receptors
…cannot cross membrane
They bind cell surface receptors
Membrane receptors for hydrophilic signaling molecules activate a wide variety of intracellular “signal transduction” pathways, including gene regulation
Most receptors for hydrophobic signaling molecules act in nucleus to regulate gene transcription
ECB 16-9
Transcription
Transcription
Intracellular signals
Intracellular receptors Cell surface receptors
A few examples of hydrophobic hormonesECB 16-11
Responses mediated by a conserved family of “steroid” receptors
HO
OH
Estradiol
OH
O
Testosterone
HO O COO-CH2 C
H
NH3+
II
II
Thyroid hormone
HO
Cholesterol
CH2OH
O
HO
C=O
OH
Cortisol
(not hormone)
Responses to hydrophobic hormones are mediated by intracellular receptors
ECB 16-12
Transcription
Translation
Cytoplasm
Nucleus
Nuclear envelope
Plasma membrane Lipophilic hormone carried
in blood
Hormone binds intracellular receptor inducing receptor dimerization and activation
Complex is imported into nucleus
Binds to “hormone response element” to regulate gene expression
Intracellular receptor
Promoter Target gene“Hormone response element”
Target cell
Receptor G-protein(inactive)
Target(inactive)
G-protein linked receptor
Cell-surface receptors - three classes
ECB 16-14
Receptor(active)
G-protein(active)
Target(inactive)
Signaling ligand
Signaling ligand
Catalytic domain(active)
Signaling ligand
Ions
Catalytic domain(active)
Enzyme-linked receptor
Ion channel-linked receptor
Receptor(active)
G-protein(active)
Target(active)
Activation of surface receptor can cause fast (cytoplasmic) or slow (transciptional) changes
Review: phosphorylation and GTPases as molecular switches
ECB 16-15
ADP
ATP Pi
PhosphataseKinase
Pi
GAPGEF
GTP
On
P
On
Energy (in the form of ATP or GTP hydrolysis) used to activate (or inactivate) signaling molecules
Energy use allows transient, high affinity/specificity interactions
Signaling with GTPasesSignaling with phosphorylation
Signal in Signal in
Signal activates protein kinase
Signal activates GEF
Signal out
Signal out
GTP
GDP
GDP
Off
Signaling GTPase
Off
Signaling protein
“Heterotrimeric G-proteins” mediate many cell signals
GDP
See ECB 16-17
G, G subunits
G binds guanine nucleotide
Receptor acts as GEF, activating G-protein
Activated G- and G regulate targets
G inactivated by GTP hydrolysis, subunits reassociate
GTP
+
GTP
GDPPi
G
(inactive GDP form)
ActiveG and G
(GTP form)
Heterotrimeric G-proteins
Downstream targets
Multiple G-proteins with distinct -, -, and -subunits (>20 known)
“Gs” stimulates or activates effectors
“Gi” inhibits effectors
“Gq” mediates Ca2+ signaling
G-protein –GDP(inactive)
GDP
Plasma membrane
Cytoplasm
Extracellular space
See ECB 16-16
“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane
receptors
Inactive receptor
Seven transmembrane domains (-helices)
Extracellular ligand-binding domain (N-terminal)
Cytoplasmic “effector” domain
Activated receptor acts as GEF to activate “heterotrimeric G-protein”
Ligand binding domain
Effector domain
1 2 3 4 5 6 7
Seven-pass
receptor
“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane
receptors
ECB 16-18 thru 16-18
Binding of ligand activates receptor
G-protein –GDP(inactive)
Inactive target
GDP
GTP
Active receptor
“Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane
receptors
Binding of ligand activates receptor
Heterotrimeric G-protein binds activated receptor
Activated receptor acts as GEF for heterotrimeric G-protein
Activated components (- and /-) regulate downstream targets
GTP hydrolysis inactivates G-protein, subunits reassociate (switches off)
Activated target
GTP
GDP
ECB 16-18 thru 16-18
Activated target can be enzyme that makes “intracellular messenger”
ECB 16-20
Ephinephrine (adrenaline) acts via heterotrimeric G protein and cAMP (intracellular messenger)
Activated adenylate cyclase forms cAMP
cAMP activates protein kinase A (PKA)
PKA enters nucleus and
phosphorylates a gene regulatory
protein
Result: altered transcription (slow)
ECB 16-24
Adenylate cyclase converts ATP to 5’,3’ cAMP
P O CH2
O
OH OH
1’
2’3’
4’
5’-O A
-O
O
P O P O P O CH2
O
OH OH
1’
2’3’
4’
5’-O A
-O-O-O
OOO
P
O
OH
1’
2’3’
5’
O
A
-O O
CH2
O
PPi 2Pi
ATP
Adenosine 3’,5’ cyclic monophosphate
(cAMP)
cAMP phosphodiesterase
“Adenylate cyclase”
AMP
Methylated xanthines (caffiene, theophylline, and theobromine ) inhibit cAMP PDE
ECB 16-21
cAMP levels rise rapidly in response to extracellular signal
ECB 16-22Serotonin is a neurotransmitter
5 X 10 -8 M cAMP
Assay fluorescence of protein that binds cAMP
10 -6 M cAMP
G-protein coupled receptors also activate IP3 and Ca2+-mediated signaling pathways
Activate receptor acts as GEF
Activated G activates phospholipase C (PLC)
Active PLC cleaves PIP2 to IP3 and diacylglycerol (DAG)
IP3 opens Ca2+ channels in ER releasing Ca2+ to cytoplasm
DAG and Ca2+ activate protein kinase C (PKC)
Active PKC phosphorylates target proteins…
Other Ca2+-dependent responses are regulated by “Calmodulin” (CaM) and “CaM kinases”
Ca2+-calmodulin activates CaM kinases, which phosphorylate and regulate target proteins
CaM contains 4 Ca2+ binding domainsCa2+
Ca2+
Ca2+-CaM binds to regulatory domains of effector proteins (e.g. CaM kinases)
see ECB 16-27
Ca2+
Phosphorylates target proteins in cytoplasm
Inactive CaM kinase
P
Active CaM kinase
ATPADP
Autophosphorylation
Calmodulin (CaM)
Catalytic domainInhibitory domain
Cells carefully regulate “free” Ca2+ levels in their cytoplasm
[Ca2+] >1 mM
[Ca2+ ]free ~0.2 M
In a resting cell, intracellular [Ca2+]free is low relative to external Ca2+…
Ca2+ is pumped into the ER (plant vacuole)
Ca2+ is pumped out of the cell by a Ca2+ ATPase and antiport with Na+ (antiport with H+ in plants/fungi)
Intracellular [Ca2+]free may increase 10-30-fold during signaling…
Moves in through channels and is released from internal stores (mostly from the ER, vacuole)
ATP ADP + Pi
Ca2+
Ca2+
2Na+ATP ADP + Pi
Ca2+
ER
Receptor G-protein(inactive)
Target(inactive)
2. G-protein coupled receptor
Last class of cell surface receptors
Receptor(active)
G-protein(active)
Target(inactive)
Signaling ligand
Signaling ligand
Catalytic domain(active)
Signaling ligand
Ions
Catalytic domain(active)
3. Enzyme-linked receptor
1. Ligand gated ion channel
Receptor(active)
G-protein(active)
Target(active)
Many growth factors bind to receptor tyrosine kinases (enzyme-linked receptor)
Receptor binds growth factor and dimerizesKinase activity activated and receptor autophosphorylates
Signaling proteins bind phosphotyrosine, activating signaling cascades
EGF and other growth factors activate Ras signaling
“GTPase Activating Protein” (Ras-GAPs) promote GTP hydrolysis by intrinsic GTPase
Pi
GAP
GTP Exchange Factors (GEFs) promote GDP/GTP exchange
Downstream effectors
Ras found to be mutated in ~30% of human tumors!
GEF
GDP
GTP
“On”RAS
GTP
“Off”
RAS(inactive)
GDP
Active Ras activates downstream signaling proteins…
MAPKKKinactive
Receptor tyrosine kinases activate intracellular Ras signaling cascades
ECB 16-31, 16-32
P
P
P
P
P
P
DRK
MAPKKinactive
MAP kinase inactive
Ras GEF
ADP
ATP
ADP
ATP
ADP
ATP
Transcriptionfactors
POther
proteinsP
Receptor kinase(active)
Growth Factors
Downstream of Receptor Kinaseactivates Ras GEF
“Mitogen-activ. protein kinase”MAP kinase
MAP kinase kinase(MAPKK)
MAPKKK active
MAPKKactive
MAP kinase active
P
PP
Regulate gene expression and protein
activity
RAS(inactive)
GDP
RAS
GTP MAP kinase kinase kinase(MAPKKK)
Mutations in Ras signaling pathway cause uncontrolled cell proliferation: cancer
MAPKKKactive
MAPKKactive
MAP kinaseactive
P
P
P
P
P
P
DRK
ADP
ATP
ADP
ATP
ADP
ATP
Transcriptionfactors
POther
proteinsP
Receptor kinase(active)
Downstream of Receptor Kinase
P
PP
Regulate gene expression and protein
activity…
RAS
GTP
RAS(inactive)
GDP
Ras GEFGTP
The Ras pathway activates expression of G1 cyclins that stimulate cell proliferation
Constituitive activation of pathway components results in uncontrolled cell proliferation = “cancer”
Cancer causing genes = “Oncogenes”
Predict effects of Ras mutations?
Signal transduction cascades are complex and interconnected
ECB 16-38
Why?• Integration
Multiple inputs to a single response…
• Divergence
Single input to multiple responses
• Amplification
• Regulation
P
P
P
P
P
P
G-protein coupled receptors
G-protein
Adenylate cyclase
cAMP
Protein kinase A
Phospholipase C
IP3
Ca2+
Calmodulin
CaM kinase
Diacylglycerol
Protein kinase C
G-protein Adapter
Ras activator
Ras
Kinase I
Kinase II
Kinase III
Receptor tyrosine kinases
Gene regulatory proteins Cytoplasmic target proteins
Communication by direct cytoplasmic continuity between cells
Cytoplasmic bridges and cell junctions
Communication via cell junctions: some embryonic cells and/or tissues are “dye-coupled”
Membrane-impermeant dye injected into on cell passes into neighbors
Cytoplasmic coupling is limited to small molecules (<1000 Da)
100 Da
1,000 Da
10,000 Da
“Gap junctions” are responsible for cytoplasmic coupling of animal cells
Membranes of coupled cells closely apposed, separated by 2-4 nm “gap”
ECB figure 19-28 MBoC figure 19-16
Large“gap jnctn”
Common in developing embryo, cardiac muscle, liver, and lens
TEM/Freeze fracture of gap junctions reveals “plaques” of intra-membrane particles
Gap junctions are composed of “connexons” made of “connexin”
hexamers
Cytoplasm of cell #2
Cytoplasm of cell #1
“Connexon”(2 per channel)
= “connexin” x 6
ECB 21-28
Plasma membrane of cell
#2
Plasma membrane of cell
#1
Extracellular “gap”(2-4 nm)
Channel is ~ 1.5 nm (~1000 Da
cutoff)
Two connexons in register form channel coupling cytoplasm of adjacent cells
The cytoplasm of plant cells is coupled by “plasmadesmata”
Membranes continuous from cell to cell
ER continuous from cell to cell thru “desmotubule”
Limited to small molecules (<800 Da), but can open to let through 20,000 Da
Primarily (but not exclusively) formed during cell division
ECB 21-30
Nucleus
Nucleus
NucleusVacuole
Plasmadesmata
Cell wall
Cytoplasm
100 nm
Cell wallPlasma membrane of adjacent cells
Cytoplasm
Cytoplasm
Desmotubule
Endoplasmic reticulum
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