Signal Transduction I

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• Signal Transduction I
• ---To put the cell in a social context
• Signals and responses
• The questions how does a cell
• in a multicellular organism
• communicate with other cells?
• how does a plant (cell) respond
• to the constant bomardments of
• stimuli from both
• inside and outside world?

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2) The language chemical, electrical, physical
contact 3) Partners cell-cell, cell-environment,
plant-other organisms
Signaling cell target cell
4) The receptors and specificity cell surface vs
intracellular, specificity of signaling
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5) The pathways and networks molecular relays
are weaved into a network or Circuit in
the Communication Processes The processes
to bring changes into the system. The changes
will Be integrated to the response as a way to
adapt to the signals
Responses
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2. Major pathways in eukaryotes as defined by
surface receptors 1) The G- protein coupled
receptors 2) The enzyme receptors 3) Ion
channel As receptors
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1. The G-protein coupled receptors

• A number of hormones or neurotransmittors utilize
  this type of the receptors
• The receptor is a
• seven-transmembrane protein
• c) Binding of ligand to the receptor
• Triggers Interaction
• With G protein
• d) The trimeric
• G protein consists
• Of alpha, beta and
• Gamma subunits
• e) Active state is the
• GTP-binding form and
• Inactive state is the GDP form.
• f) GTP binding dissociates the 3 subunits
• And activate the G protein. GTP
• Hydrolysis into GDP inactivates it.

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• 2) HOW TO STUDY RECEPTORS?
• Binding assay to see where is the receptor, one
  can utilize a labeled ligand to do the binding
  assayyou can fractionate the cells to purify
  specific organelle of membrane to mix with the
  ligand and see if it binds to the fraction. Three
  parameters will be critical the specificity and
  affinity should be consistent with the biological
  function of the ligand. The binding should be
  saturable because there is only a limited
  number of the receptor in a fraction. Then purify
  the receptor protein and clone the genes.

total
specific
amount
Non-specific (cannot be competed by the
unlabeled ligand)
Concentration (biological?)
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B) Functional cloning of the receptor by randomly
expressing a cDNA library in a model system (eg,
oocytes or culture cells) monitor the function
of the receptor by adding the ligand and measure
specific response in the expression system.
Retrieve the specific mRNA that causes the
response
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• 3) The targets for trimeric G proteins and
  production of second messengers
• The cAMP pathway
• The beta-adrenergic receptor binds adrenaline and
  activates a G protein that in turn activates
  adenylyl cyclase that forms cAMP from ATP, cAMP
  functions as second messenger to activate a
  protein kinase called PKA, PKA phosphorylates
  glycogen phosphorylase kinase (GPK), this kinase
  then phosphorylates glycogen phosphorylase (GP)
  that degrades glycogen into glucose---blood sugar
  level increasesthe response.

glucose
glycogen
GP
ATP?cAMP?PKA?GPK
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How does cAMP work to activate PKA? What is a
second messenger?
cAMP
C
R
C
R
C
R
active
C
R
Inactive
C
R
C
R
2nd messenger small molecule that is produced by
the primary signal and functions to pass the
signal further downstream to cellular response.
b) The IP3-Ca2 pathway The trimeric G proteins
are present in many forms (different proteins
encoded by different genes). Some forms target
adenylyl cyclase, and some other forms target
other enzymes such as PLC (phospholipase C), PLC
cleaves phosphotidylinositol 4,5 triphosphate
(PIP2) and produce IP3 and Diacylglycerol (DAG),
these second messengers activate different
targets---IP3 activates a calcium channel in the
ER membrane and release calcium into the cytosol
and DAG activates a protein kinase called PKC.
Calcium can be sensed by protein sensors such as
calmodulin (CaM) that binds calcium and change
conformation, CaM then activate many other
proteins including a protein kinase that
phosphorylates myosin light chain and activate
the muscle activity.
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Calcium is a critical Second messenger in Plant
cells. It is very Tightly regulated by Many
proteins Like channels and pumps Many
organelles Are involved in Calcium homeo- stasis
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Measuring calcium levels in living cells
fluorescent dye, aequorin, cameolin (CaM-GFP),
etc Pollen tube growth and calcium wave
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How does calcium works? Calcium sensors detect
the changes in calcium concentration by Binding
it and conformational changes. Such change will
trigger Interaction with downstream targets such
as protein kinases that modify other protein
activity.
CaM takes a new structure when in calcium-bound
form. A specific sequence in CaM forms the
Ca-binding hand shown at right.
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DAGthe other second messenger produced from PLC
activity The target PKC needs calcium in order to
get associated with the Membrane where DAG
activates the kinase activity
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2) The enzyme receptors receptor kinases as
example Many growth factors in animals such as
epidermal growth factor (EGF) work by binding to
their surface receptors that are tyrosine
kinases. Binding triggers dimerization of the
receptor and activation of the kinase activity.
The kinases phosphorylate each other and produce
a highly phosphosphorlyated cluster that serves
as a module for specific interaction with the
adaptor proteins. These proteins then interact
with other proteins and pass the signal
downstream to a small G protein called Ras, one
of the earliest oncogenes to be discovered. Ras
activates Raf, a ser/thr kinase that activate
MEK, another kinase, that activates MAPK, still
another kinase. MAPK activates proteins that
activate gene expression---response. See the
picture next page.
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How phosphorylation changes MAPK structure (spot
the major difference of these two structures)
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How does MAPK cascade determine the specificity
of signals? --The scaffold proteins hold the key

• Different isoforms in each step of the relay
• Different scaffold proteins recruit
• Different combination of the relay molecules
• c) Different signal and outcome

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3) RLKs in plants---a large family of receptor
proteins (lt700) RLKs (Receptor-Like Kinases)
contains an extracellular domain, a
transmembrane domain, and a cytoplasmic kinase
domain It is hypothesized that RLKs work Like
tyrosine kinase receptors in Animals and form
dimer after ligand Binding. Kinase becomes
activated and phosphorylates each other to form
A binding module to recruit other Proteins onto
the membrane. It is also believed that MAPK
cascade Is downstream from RLK.
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• The apical meristem development involves a RLK
  pathway
• Genetics analysis of meristem size identified
  mutants that produce larger meristems and more
  flowers. One group of mutants called clavata 1
  are mutated in a RLK gene, clavata 2 is mutated
  in a RLK-like gene (everything like RLK but
  without the kinase domain), the third mutant is
  mutated in a gene encoding a small protein
  localized to the cell wall. Further study showed
  that CLV1/CLV2/CLV3 are present in a
  supermolecular complex in the plasma membrane.
  This complex also recruits other proteins
  including a protein phosphatase (KAPP), and a
  small G protein Rop. Rop may be like Ras that
  pass the RLK signal down to MAPK cascade.

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WT clv1
Second example is stomatal development that
appears to be controlled by RLK as well. Third
example is the symbiotic interaction RLK serves
as receptor for a nodulation factor from
bacterial cells. Fourth, you already heard
about WAKs and their role in cell expansionthey
belong to RLK super-family also. The list will go
on and on as more work is done with the
functional analysis of the RLK genes. Friday
lecture will give you at least one more example
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4). From cell surface to the nucleus cAMP
pathway and receptor kinase pathway not only
would modify the cellular components in the
cytoplasm but in many cases they can lead the
signaling processes into the nucleus to modify
gene expression.
PKA catalytic Subunit (c) move into the nucleus
and phospholrylates the transcription factor CREB
that forms protein complex with P300 and turns on
gene transcription
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Similarly, the MAPK can be transported into the
nucleus after phosphorylated by MAPKK. In nucleus
it also phosphorylate some transcriptional
proteins for gene activation.
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5) Signals must be removed after
activation --Cell signaling is defined by
temporal and spatial information. It is not
always on but rather have frequencyon and off,
on again and off again To keep a frequency,
off is equally importantthe signal must be
removed. --Very often the signal is not present
but arrives when needed---so when the signal is
not there anymore, the pathway will be shut down.
This shutting down process also involves
removal of active molecules. Simplest and
could be most important example kinase and
phosphatase work in pairs to modify the protein
structure/function by putting on or taking off
the phosphate from the protein. Another one is
the calcium elevation in the cytosol and removed
by sequestering it into the vacuole or other
organelles. Third one is the G protein switch
GTP vs GDP binding forms
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3. Crosstalk and signaling networks Pathways in
the cell interact with each other and regulate
each other. Therefore, the linear pathways are
weaved into a complex network like a public
transportation network that consists of buses,
subways, and trains A component in the
network can be considered as a knot on the
network (a fishnet). All components are connected
to each other directly or indirectly. Anything
wrong with one component may affect the status
of other components.
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An example of pathway Interaction Protein
kinases in the G- Protein coupled receptor
Pathway can regulate the Activity of MAPK and
Vise versa. The kinases could also Share a
common protein As substrate so the path- Way may
converge at this Protein substrate.
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• 4. Evolution of signaling pathways from bacteria
  to human
• The two component system dominates the bacterial
  world
• The signal receptor and response
  regulator---phosphate transfer from histidine to
  aspartate

2) This system was passed on to plants and fungi
but not animals The histidine kinase and the
response regulators have fused together in many
cases although some separate response regulators
still exist in fungi and plants.
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3) Many new paradigms appeared in eukaryotic
systems Think about the complexity of the cell
structurefrom prokaryotes to eukaryotes The
G-protein coupled receptors dominate the animal
signaling, but very few pathways in fungi and
plants use this. The tyrosine kinase receptors
have never been found in fungi and plants! Plants
developed a large number of ser/thr receptor
kinases that are not a major theme in animals.