Cell Membrane II: Cell Signalling

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Cell Membrane IICell Signalling

• Dr. Bill Diehl-Jones

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Agenda

• Communication Modes and Mechanisms
• Short vs. Long Distance
• Intercellular Signaling Mechanisms
• Signaling systems
• positive/negative feedback, feedforward

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CommunicationModes and Mechanisms
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Modes of Communication

• Long Distance
• Endocrine
• Nervous
• Neurendocrine

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Modes of Communication

• Short Distance
• Autocrine and Paracrine
• Direct Link
• Gap Junctions

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Intercellular Signaling Mechanisms

• Direct gating of ion channels
• Extracellular ligands

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Extracellular Ligands

• Lipophilic signals cross the plasma membrane
• Eg steroid hormones (testosterone, estrogen,
  aldosterone)
• Receptors are intracellular
• Lipophobic signals do not cross the membrane
• Eg

Intracellular Receptors
Extracellular Receptor
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Once a ligand binds to a receptor, stuff
happens...
Ligand
Y
G
A
Receptor
? Cai
X
Z
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Lipophobic Signals

• Extracellular signals are transduced and
  amplified
• Transduction
• Process by which a signal crosses the membrane
• Amplification
• Process by which the effect of a signal is
  multiplied

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Transduction

• Occurs one of two ways
• via direct phosphorylation

Ligand (eg a hormone)
Protein
Receptor
Protein
Phosphate Group
An example we will discussRTK Receptors
Or...
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Transduction

• A far more common form of transduction is via G
  proteins ...

A
G
Amplifying Enzyme
G protein
Ligand
Receptor
We will focus on these types of mechanisms
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Amplifyng Enzymes

• Different flavours
• cAMP, cGMP, Phospholipase C. IP3
• Activate second messenger cascades

Amplifying Enzyme
1st Order Second Messenger
2nd Order Second Messenger
3rd Order Second Messenger
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So What?

• Steps in the cascade allow amplification of the
  signal
• A few hormone molecules activate numerous
  enzymes.
• e.g. each PKA molecule (a second messenger) can
  activate several phosphorylase kinase molecules.
• PKA has three different and coordinated effects.
  Effects - key to coordination of separate
  responses by the cell

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Focus on G Protein-Mediated Signaling
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G-Protein Coupled Receptors

• Many hormones work through GPCRs
• As many as 2000 different GPRCs
• many not yet known genes identified
• no known ligand yet
• Caenorhabditis
• 1/20 of all the proteins are GPCRs
• Humans
• one quarter of all prescription drugs work
  through GPCRs

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G-Protein Coupled Receptors

• A family of integral proteins, all have seven
  trans-membrane a-helix segments
• All work in the same way
• through the Heterotrimeric G-proteins
• turn on effector molecule which makes the second
  messenger
• epinephrine and glucagon (e.g.) turn on adenylyl
  cyclase to make cAMP 2nd messg.
• others (e.g. acetylcholine) use phosphoinositol
  , DAG second messengers
• others (e.g. photoreceptors) use cyclic GMP

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G protein action

• 1. When receptor combines with ligand, receptor
  changes shape and binds the a subunit of the G
  protein
• 2. Activation of the receptor a subunit then
  exchanges a GDP for a GTP, entering activated
  state
• - the receptor/ligand can activate several G
  proteins, as long as ligand is bound
• 3. Relay the a subunit dissociates from b,g and
  associates with effector, producing second
  message
• - b,g stay together
• - second message is made for duration of binding

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G Protein Action
receptor
ligand
g
a
b
1.
GDP
Heterotrimeric G-protein
GTP
2.
GDP
exchange
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G protein action
g
a
Effector. (such as adenylyl cyclase or PI-PLCb)
b
GTP
3.
Second Message
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G-proteins Have Different Effects

• Four types of G-proteins, different Ga subunits.
• Gas stimulates adenylyl cyclase
• Gaq activates PLCb
• Gai inactivates adenylyl cyclase
• Ga12/13 activates Src, Ras, phospholipase D and
  protein kinase C
• The Gbg complex can also activate other
  effectors.

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Integrated Example

• Glucagon and Epinephrine Signaling

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A word from our sponsors glucagon and
epinephrine effects on glucose

• Utilization of glucose
• primary energy source
• stored as insoluble polymer, glycogen
• glycogen to glucose is promoted by hormones
• glucagon (released from pancreas), boosts blood
  glucose
• epinephrine (adrenal gland), boosts blood glucose
  during stress
• Promote breakdown of glycogen to
  glucose-1-phosphate, first step in catabolism
• Glucose is either catabolized or sent to
  bloodstream for delivery to other places.
• Inhibit glycogen synthase, this enzyme makes
  glycogen, so it has to be turned off in order for
  the cells to release or burn glucose.

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The Process

• 1. Hormone binds to receptor and heterotrimeric
  G-protein
• 2. Activation of effector Adenylyl cyclase,
  formation of cAMP, diffuses into cytoplasm
• 3. cAMP binds to allosteric site, activating
  Protein kinase A (PKA)
• 4. This phosphorylates target Glycogen
  synthase, inactivating it. Glycogen no longer
  produced
• 5. At same time, phosphorylates enzyme
  Phosphorylase kinase, activating it

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The Process contd

• 6. the phosphorylase kinase then phosphorylates
  target enzyme Phosphorylase, activating it,
• 7. phosphorylase catalyzes glycogen break-down,
  glucose-1-phosphate is released
• 8. also at same time, in the nucleus, PKA
  phosphorylates transcription factor cyclic AMP
  response element binding protein (CREB)
• 9. phosphorylated CREB dimerizes and binds to
  cAMP response element (CRE), turning on PEPCK
  gene, gluconeogenisis increases

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Glucagon, Epinephrine
G
adenylyl cyclase
active
inactive
Phospohdiesterase
cAMP
ATP
Glycogen Synthase
active
Breakdown of glycogen into glucose-1-phosphate
inactive
active
Protein Kinase A
P
P
inactive
Stops making glycogen
active
Phosphorylase
inactive
Phosphorylase Kinase
Effect in Nucleus
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Glucagon/Epinephrine Action contd
Nucleus
Cytoplasm
CREB plus ATP
cAMP
cAMP
active Protein Kinase A
Pepck gene
CREB
CRE
PEPCK enzyme Starts making glucose
DNA
PEPCK mRNA
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Termination of the signal

• cAMP is broken down by phosphodiesterase
• Phosphatase reverses the phosphorylation of the
  three proteins
• phosphorylase kinase
• glycogen synthase and
• phosphorylase
• Adenylyl cyclase remains active while hormones
  are present. The cell has a way to stop adenylyl
  cyclase activity when it the hormones are
  removed.
• How?...

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Termination (the steps)

• a subunit is a GTPase, which hydrolyzes GTP to
  GDP and inactivates itself
• inactive a subunit reassociates with b and g
• Requires an additional factor RSG
• RSG enhances GTPase and speeds up the timing step
• this causes a drop in ligand concentration,
  resulting in
• dissociation and inactivation of the receptor

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Another Means of Termination

• Active inactivation of the receptor, a 2 step
  process of desensitization. This means that the
  cell stops responding, even when ligand is still
  present around the cell
• 1. Phosphorylation by G-protein receptor kinase
  (GPRK), inactivates the receptor,
• 2. The phosphorylated receptor binds another
  protein called arrestin, which acts as adaptor
  for Clathrin, allowing receptors to be
  internalized, thus further desensitising the cell

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The Big Picture
g
a
2 ATP
P
b
stops
P
GDP
GRK
2 ADP
Pi
Arrestin
RSG
- G-protein hydrolyzes GTP, inactivates itself -
receptor is phosphorylated, becomes inactive -
phosphorylated receptor binds arrestin, an
adaptor for endocytosis
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Lipid Second Mesengers
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Lipid Second Messengers

• GPRC intracellular messengers made by
  phosphorylation and hyrdrolysis of the membrane
  glycerophosphate phosphatidyl inositol

inositol sugar phosphate glycerol fatty
acids
- P -
kinases
- P -
P
P
phospho inositol 4,5, biphosphate (PIP2)
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Lipid Second Messengers

• Produced by phoshatidylinositol-specific
  phospholipase C.
• abbreviated PI-PLCb
• produces two signal molecules
• Diacylglycerol (DAG), which stays within
  membrane
• and Inositol triphosphate (IP3), highly soluble,
  enters cytoplasm

P
O

P
-O - P - O -
O-
DAG
IP3
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One Exampleacetylcholine effects on a smooth
muscle cell

• 1. inositol phosphate is phosphorylated.
• phospho inositol 4,5, biphosphate (PIP2)
• 2. acetylcholine binds to plasma membrane
  receptor
• 3. G protein system is activated (Gaq)
• 4. the phospholipase PI-PLCb is activated by the
  G protein
• 5. PIP2 is metabolized to diacylglycerol DAG and
  inositol phosphate IP3

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• 6. DAG in turn, stimulates Protein Kinase C which
  acts to promote muscle contractility,
  phosphorylates elements of the actin/myosin
• 7. the IP3 binds to the SER membrane, to IP3
  receptors
• 8. IP3 receptors are calcium channels, they
  release calcium from SER
• 9. Calcium ion concentration of cytoplasm
  increases
• 10. muscle cell contracts
• 11. IP3, DAG are rapidly degraded, calcium is
  rapidly pumped back to SER

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Phosphatidylinositol Second Messengers
acetylcholine
PI-PLCb
G
DAG
PIP2
active
muscle cell contraction
Protein Kinase C
IP3
increased Ca
increased muscle contractility
IP3 receptor (a ligand-gated chanel)
smooth endoplasmic reticulum
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The same basic systems are used to activate
different responses to different signals

• a. IP3
• i. Vascular smooth muscle contraction
• ii. smooth muscle contraction
• iii. skeletal muscle contraction
• iv. blood platelet aggregation of platelets
• b. Protein Kinase C. cell growth,
  differentiation (development into different
  tissue types), metabolism.
• i. blood platelets serotonin release
• ii. mast cells histamine release
• iii. smooth muscle contractility
• iv. nerve cells neurotransmitter release
• v. adipose tissue fat synthesis
• vi. liver cells glycogen hydrolysis

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• c. cAMP (and PKA) in different cells
• i. liver epinephrine -glycogen hydrolysis,
  glucose synthesis, and glucagon -reduction in
  glycogen production
• ii. kidney vasopressin - activation of aquaporins
• iii. thyroid cell TSH - thyroid hormone release

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The Other Receptor ClassReceptor Tyrosine
Kinases (RTKs)

• Receptor is an enzyme
• a protein tyrosine kinase which phosphorylates
  proteins at tyrosines
• Eg
• insulin receptor
• growth factor receptors
• MAP kinase cascade

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gt 50 Types of RTK Receptors

• Related receptor molecules
• Similar structure
• Similar method of action, but
• different ligands
• different downstream effects.
• all have a single trans-membrane segment
• Involved in regulation of
• Growth (epidermal growth factor, platelet derived
  growth factor, insulin).
• Cell division (disorders of RTK receptors lead to
  uncontrolled cell division, cancers).
• Cell survival/death
• Attachment of cells to extracellular matrix
• Migration of cells

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An Example Insulin receptor action through the
RTK type receptor

• Insulin is produced when blood glucose increases
  after eating.
• It causes glucose uptake by liver, muscle, fat
  cells
• Acts to decrease blood sugar, cells produce
  glycogen or fat, gluconeogenisis is inhibited.

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Mechanism of Action

• 1. Receptor binds ligand on exterior of the cell.
• the insulin receptor is a tetramer, two
  extracellular alpha chains with ligand binding
  sites and two beta chains with kinase activity,
  all are linked by disulfide bridges.
• 2. Activation of the receptor
• insulin receptor is unusual in existing in dimer
  state even before binding the ligand
• tyrosine kinase activity, trans-autophosphorylati
  on.
• it also phosphorylates another protein, Insulin
  Receptor Substrate (IRS).
• The phosphorylated IRS stays on the insulin
  receptor by a PTB domain

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Mechanism of Action contd

• 3. Other proteins recognize, bind and are
  activated by the phosphorylated tyrosines on
  receptor and IRS
• 4. final actions of insulin
• 1. PI PLCg eventually (via the second messengers
  IP3, Diacylglycerol, Protein kinase C (PKC), and
  calcium, described in previous lecture) causes
  cell proliferation
• 2. Protein Kinase B PKB causes
• glucose uptake into cells by transferring the
  GLUT 4 transporter in muscle and fat cells.
• increased protein synthesis
• stimulation of glycogen synthase
• 3. Ras Promotes protein synthesis, growth,
  proliferation

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Insulin receptor
insulin
1. receptor - ligand binding
binding site
a
a
b
b
2. tyrosine kinase activity - auto -
kinase - Insulin receptor substrate
a
a
b
b
P
P
P
P
P
P
P
P
P
P
IRS1
IRS1
P
P
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Insulin Receptor Action...
a
a
b
b
P
P
P
P
P
P
P
P
3. effectors with SH2 domains bind to
phosphorylated tyrosines
P
activate PI-PLC g
P
IRS1
phosphatidyl inositol 3 hydroxykinase
P
P
GDP
PI3K
Grb/Sos
activate RAS
activate Protein Kinase B
GTP
Glucose transport, glycogen synthesis
actions
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Other RTKs the growth factors
1. some tyrosine kinases exist as monomers which
come together only after binding ligand. Then
they start the tyrosine kinase activity. 2. the
growth factors, PDGF (platelet-derived), EGF
(epidermal), FGF (fibroblast). 3. These
activate the MAP Kinase cascade - including a GTP
binding protein Ras (Ras is a non-trimer G
protein they are not called G protein coupled
receptors). Ras is in active state when bound to
GTP. When active it activates downstream
effectors. 4. Cause tyrosine phosphoylation by
tyrosine kinase located on inner membrane of the
receptor protein (or in case of insulin,
phosphorylate the IRS).
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Calcium as a Second Messenger
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Calcium as second messenger

• we just saw calcium acting as an intermediate of
  IP3 action in the cell, but that is not the only
  way it is activated
• it can also be released in response to calcium
  channels on the cell surface Calcium dependent
  Calcium release
• in this case calcium is both the first and the
  second messenger
• this accomplishes rapid amplification and rapid
  coordination

primary Ca channel
S.E.R.
Ca
secondary Ca chanel
Ca
effect
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Types of Calcium Responses

• voltage dependent channels in nerves, some
  muscles
• receptor-mediated calcium channels
• fertilization, calcium channels open up in
  oocytes following fertilization.
• prevent further sperm entry anywhere around the
  large cell (rapid, long-distance)
• begins activation of cyclins, starting cell
  division (coordinating effect)
• Plants open the outside calcium channel in
  response to light, pressure, plant hormones
  (abscisic acid)
• calcium is stored in the vacuole of plant cells

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calcium-induced calcium release
1.
10-3 mol/l
calcium chanel
6. one effect is to promote calcium removal
4. released Ca binds to calmodulin
resting ca concentration 10-7 mol/l
5. effects
2. Ryanodyne receptor
3. release
smooth endoplasmic reticulum or plant cell vacuole
high Ca conc. 10-3 mol/l
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The Steps

• 1. Calcium ions enter cell through activated
  channel
• 2. they bind to the Ryanodyne receptor.
• it does the same thing as the IP3 receptor, but
  it is different. it does not respond to IP3
• 3. caclium ions released from the smooth
  endoplasmic reticulum (or the vacuole in plants)
  diffuse down concentration gradient, diffuse
  throughout cell
• 4. calcium ions bind to calmodulin
• 5. intracellular effects
• 6. one of the effects is to pump calcium back out
  of the cytoplasm to s.e.r, the plant vacuole and
  the exterior of cell termination of signal.

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Why are there multiple steps?

• 1. amplification of signal. (exponential).
• e.g. one receptor can turn on several
  heterotrimeric G-proteins before inactivation,
  each G-protein can activate several effector
  molecules before inactivating itself
• 2. coordination
• a. spatial coordination throughout the cell by
  diffusion of second messengers.
• cAMP,
• Ca are small molecules, diffuse fast, works
  well in large cells such as muscle, fertilized
  egg, neuron
• b. coordination of different responses a number
  of different enzymes have to work together to
  accomplish metabolic pathways.

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Interconnections of signaling pathways

• 1. Convergence. two receptors cause the same
  signal to be activated.

EGF
acetylcholine
PIP2
PI PLCb
PI PLCg
IP3
DAG
common effects
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Interconnections of signaling pathways

• 2. Divergence. one ligand has several effects
• - e.g. the insulin receptor (through IRS protein)
  activates
• Ras
• PI-PLCg
• Protein kinase B (PKB)
• e.g. Protein kinase A (PKA of the
  glucagon/epinephrine receptor), turns on three
  different proteins

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Interconnections of signaling pathways

• 3. Crosstalk, pathways interconnect (a bit of a
  case of convergence)
• e.g. every one of the intermediate steps in the
  map kinase cascade can affect more than one
  substrate.
• CREB activation (see fig 15.33)
• epinephrine activates PKA which enters the
  nucleus when activated by epinephrine,
  phosphorylates CREB
• EGF activates Raf, which activates MAP Kinase
  which also phosphorylates CREB
• Crosstalk PKA inhibits Raf. blocking the
  effect of EGF on CREB

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So What?

• cells can keep signals separate
• second messengers diffuse rapidly, but some
  steps, including the kinases (such as PKA) are
  kept in certain locations in the cell.
• PKA is actually held in place by special binding
  proteins (called AKAPs), focusing its effect in
  the cell.
• there is evidence that the steps in the MAP
  Kinase cascade are kept together in one location
  in the cell by special scaffolding proteins,
  limiting the location of the downstream cell
  response.