AH Biology: Unit 1
Cells and Proteins
Membrane Proteins:
Signal Transduction
Signal transduction
•
Where extracellular messages are hydrophilic, eg protein hormones such as STH
(human growth hormone) and insulin or neurotransmitters such as acetylcholine, they
are unable to pass directly through the lipid bilayer.
•
They are not directly transported across membranes, but instead their message is
transduced, ie converted into an intracellular signal by the act of binding to target
receptors in the plasma membrane.
•
We are going to consider, using case studies, three such systems at the cell surface:
–
–
–
ion-channel-coupled receptors, eg ligand-gated ion channels
G-protein-coupled receptors
enzyme-coupled receptors.
Ion channel coupling
•
The next slide illustrates how an action potential travelling along one nerve cell can
be transmitted to another.
•
The junctions between nerves or nerves and muscles are called synapses.
•
The transduction of the message is required.
•
The action potential initiates the fusion of neurotransmitters containing vesicles with
the presynaptic membrane. These chemicals diffuse across the synapse, triggering
ion-gated Na+ channels on the post-synaptic membrane, which initiates the
generation of an action potential in the target cell. This may result in action potential
propagation down the nerve or the triggering of myofibril contraction in
neuromuscular junctions.
•
Neurotransmitters are rapidly reabsorbed after release or broken down.
•
This class of transduction is regulated by controlling the influx and efflux of ions with
associated voltage-gated or ligand-gated channels.
Ligand-gated ion channels
+
Na
Na+
Na+
+
Na+
Na
Initial influx of Na+ depolarises the
membrane. This may trigger local voltagegated channels to open and cause a further
influx of Na+. Such rapid depolarisation
spreads to other Na+ channels, creating an
action potential that spreads to the entire
plasma membrane.
G-protein-coupled signal transduction
G-protein coupled
•
In this system, G-protein-coupled receptors (GPCR) activate a second protein, the Gprotein.
•
GPCRs have seven transmembrane alpha-helix domains.
•
The G-protein is a guanine triphosphate (GTP) binding protein and is bound into the
intracellular face of the plasma membrane.
•
On activation by the GPCR, GTP binds to and activates the G-protein complex.
•
The G-protein then activates a separate target protein. This is usually an enzyme or ion
channel.
•
If the target protein is an enzyme, activation may start a cascade of small intracellular
signalling molecules within the cytoplasm, triggering intracellular events.
•
If the target protein is an ion channel, activation may result in the mediation of plasma
membrane permeability.
G-protein-coupled signalling
G-protein-coupled signalling case study
Production of cyclic AMP (cAMP)
•
Some classes of GCPR stimulate the production or inhibition of cAMP by adenyl
cyclase.
•
This makes cAMP from ATP in the cytoplasm. This molecule is a potent intracellular
signal. It should be noted that there are also GCPR-linked systems whose role is to
reduce cAMP levels.
•
Hormones such as adrenaline, glucagon and thyroid-stimulating hormone (TSH) act
on their appropriate receptor, triggering G-protein and switching on adenyl cyclase,
which is plasma membrane bound, raising cAMP levels.
•
cAMP is destroyed by cAMP phosphodiesetrase.
G-protein-coupled signalling case study
•
cAMP can activate a second group of enzymes called cAMP-dependant kinases.
•
Once bound to cAMP these kinases are activated, releasing active catalytic subunits.
These can diffuse through the nuclear pores where, for instance, they can
phosphorylate gene regulatory proteins called cAMP response element binding
proteins (CREBs), which can then stimulate gene transcription.
•
Raised cAMP can also trigger some of the effects of the following ligands when
stimulated through the appropriate GCPR/G-protein activation.
– Adrenaline is a hormone that has many different effects depending on what
target receptor it stimulates. Many different classes of adrenaline receptor exist
and depending on what type the target cell expresses the effect may be very
different. For example, myocardial tissue in the heart will increase the rate and
contraction of its cells when triggered by adrenaline. Adipose cells (fat cells) are
triggered to break down triglycerides and release fatty acids and glycerol into the
bloodstream.
– TSH acts on thyroid cells, stimulating thyroid hormone synthesis and secretion.
– Glucagon stimulates liver cells to break down stored glycogen to glucose.
G-protein-coupled signalling case study
Cascades
• At each step the generation of multiple linked intracellular messages creates
interlinked signal systems called cascades, eg molecule 1 stimulates production of
molecule 2 etc.
•
Cascades can greatly magnify the response. Initial binding of one signal molecule to
its GPCR can generate many copies of intracellular signal molecules such as cAMP
or IP3.
G-protein-coupled signalling case study
An example of ion channel regulation by G-protein coupled signalling
•
Acetylchoine, a
neurotransmitter, can
reduce cardiac muscle
activity.
•
Efflux of K+ balances
influx of Na+ into
cytoplasm from
outside, and Ca+ from
intracellular
compartments.
•
This makes it more
difficult for the target
membrane to
depolarise and trigger
cell contraction.
•
These acetylcholine
GPCR are different to
the ligand-gated
channels found at
neuromuscular
junctions in skeletal
muscle and nerve-tonerve synapses.
G-protein-coupled signalling case study
Regulation of the cytoskeleton by G-protein-coupled signalling
•
Two subfamilies of G-proteins, G12/G13 , are involved in cytoskeletal remodelling by
promoting actin–myosin contraction.
•
An associated family Gi is involved with actin fibre polymerisation and membrane
protrusion.
•
Two pathways exist. Stimulation of the G1-linked receptor stimulates the Rac pathway via
the production of the intracellular signal for phosphatidyl inositol triphosphate (PIP 3), which
induces polymerisation of actin filaments and the extrusion of the cell towards the stimulus.
•
Simultaneously, stimulation of the G12/G13 linked receptor stimulates the Rho pathway,
causing actin–myosin contraction.
•
All the intermediate messengers are short lived so the stimulus is polarised to the surface
in contact with the stimulating ligand.
•
The Rho and Rac pathways appear to be antagonistic so if Rac is stimulated at the front
Rho can only be stimulated at the rear. This allows directed movement towards a stimulus
with contraction of cytoskeleton behind and extrusion at the front.
•
The movement of neutrophils towards bacteria has been characterised in this manner.
G-protein-coupled signalling case study
Olfactory sensitivity
•
Olfactory receptors in animals are good examples of GPCRs that are sensitive to
odours.
•
The receptors are localised on modified cilia protrusions from the olfactory neurons.
•
Different classes of olfactory sensors exist for many different molecules. Each is
encoded by different genes. What they all have in common is their mode of action.
•
Each GPCR activates an olfactory G-protein, which in turn stimulates the production
of cAMP by adenyl cyclase.
•
Increased cAMP levels stimulate associated cAMP-gated Na+ channels, resulting in
an influx of Na+ ions depolarising the target membrane. If depolarisation is strong
enough an action potential is set up that causes the nerve to fire and send the action
potential down the axon, where it generates a nerve impulse. These impulses send
sensory information about smell to the central nervous system.
G-protein-coupled signalling in
photoreception
•
Vertebrate vision is mediated by photoreceptors coupled to G-proteins.
•
The photoreceptor is coupled to a G-protein that switches off production of GMP
cyclase and results in falling cGMP. This closes associated cGMP-gated cation
channels and results in reduced neurotransmitter release from associated neurones
when the photoreceptor is illuminated.
•
cGMP-gated cation channels are kept open by high levels of cGMP, inhibiting
depolarisation. As cGMP levels fall these channels close.
•
The postsynaptic neurones lose their inhibition and an action potential is generated
along the neurone as depolarisation is promoted. The neurone is said to be excited. A
nerve impulse is then sent along sensory nerves to the CNS.
•
Light detection and its magnification are discussed in detail in a separate
presentation.
Enzyme-coupled signalling
In this form of signal transduction
the binding of a ligand stimulates a
transmembrane enzyme directly.
These molecules span the bilayer,
existing singly or as two proteins
stimulated by dimers.
Ligand binding stimulates an
enzyme directly rather than via a
G-protein.
The cytoplasmic portion containing
the catalytic site may have
additional domains.
Alternatively, a transmembrane
receptor in permanent association
with an enzyme undergoes a
conformational change on ligand
binding which results in enzyme
activation.
Enzyme-coupled signalling case study
Receptor tyrosine kinases (RTKs)
•
Many enzyme-coupled signalling systems fall into these families.
•
On stimulation by their appropriate ligand they are able to auto-phosphorylate
tyrosine amino acids on their cytoplasmic domains through the activation of their
catalytic site.
•
The phosphorylated tyrosines may in turn provide binding sites for intracellular
signals, which are then activated themselves by becoming phosphorylated or by
undergoing a conformational change through binding to the phosphorylated tyrosine
residues.
•
In turn this may start an intracellular cascade by activated intracellular signals,
including the stimulation of some of the same intracellular signals as G-protein-linked
systems, eg phopholipase-C-linked cascades.
•
Once stimulated and the signal transduced, the receptor–ligand complex is
internalised by receptor-mediated endocytosis, switching off the response. The
recycled receptor minus ligand is then returned to the plasma membrane.
Enzyme-coupled signalling case study:
insulin
–
This hormone triggers many intracellular messengers. Binding to its receptor stimulates
phosphorylation of its receptor and associated signal molecules, triggering PIP3 production
and its associated cascade. This gives rise to expression of glucose transporter Glut 4 on
the plasma membrane, increasing intracellular glucose levels. Additionally, glycolysis and
fatty acid synthesis are promoted, as is glycogen synthesis.
–
Diabetes mellitus is a condition associated with impaired insulin function either through
insufficient insulin production (type 1/insulin dependant) or insulin resistance related to
insensitivity or density of insulin receptors (type 2/non-insulin-dependant, NIDDM).
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Some rare mutations of insulin receptors can give rise to conditions such as Donohue
syndrome (leprechaunism). In this case the receptors lose their affinity for insulin because of
an inherited recessive mutation, giving rise to complete resistance to insulin. This condition is
universally fatal, with most sufferers dying within 2 years of birth.
–
Other mutations have been identified which relate to reduction in insulin sensitivity by
mutations retarding the ability of the cell to recycle the receptor back to the plasma
membrane. This may provide a partial model for loss of insulin sensitivity in type 2 (NIDDM)
diabetes.
Enzyme-coupled signalling case study
Insulin action
Effect of insulin on glucose uptake and metabolism
1. Insulin binds to its receptor, resulting in activation of tyrosine
kinase.
2. Activation of intracellular message cascades trigger the following:
• glut-4 transporter to the plasma membrane and influx of glucose
• glycogen synthesis
• glycolysis
• fatty acid synthesis.
Enzyme-coupled signalling case study
Cell growth factors
Examples: epidermal growth factor (EGF), fibroblast growth factors (FGFs)
•
Most cell growth factors stimulate cell proliferation and cell growth through increasing
mitosis and DNA activity.
•
The genes encoding the growth factor and their receptors are mainly protooncogenes as mutation of the same can give rise to oncogenes, which promote
uncontrolled cell division.
Enzyme-coupled signalling case study
Epidermal growth factor
• Coded for by a proto-oncogene.
• Its receptor EGFR is an RTK which is also
coded for by a proto-oncogene.
• The diagram shows the multitudinous ways
that the stimulated receptor homo dimer
can generate intracellular signals and their
potential outcomes.
• One effect of EGFR stimulation is the
transcription and expression of EFGR
genes.
• Over-expression of EFGR and EGF is
linked to cancer and is an expression of
oncogenic mutations.
Enzyme-coupled signalling case study
Fibroblast growth factors
•
This family of ligands is involved in the control of cell growth and cell differentiation,
vascularisation, wound healing and embryo growth.
•
They act through an associated family of RTKs called fibroblast growth factor
receptors (FGFRs) in a similar manner to EGFRs.
•
One subclass of receptor, FGFR3, has been associated with bone growth as FGFR3
stimulation seems to inhibits the formation of bone from cartilage.
•
Mutations in FGFR3 have been associated with achondroplasia, the most common
form of dwarfism (~1 in 25000 live births world wide), which results from retarded long
bone growth.
•
Achondroplasia is a non-sex-linked dominant condition in which sufferers inherit an
allele for mutated FGFR3 that is overly active.
Enzyme-coupled signalling case study
Molecular medicine
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Current advances in molecular biology are focusing on targeting specific RTKs as
those related to cell growth factors are often over-expressed in cancer cells.
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Drugs and monoclonal antibodies are in development to try and block such receptors.
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Herceptin® and Erbitux® are commercial monoclonal antibodies that block two types
of EGF receptor (Her-1 and Her-2) that can be over-expressed in some types of
breast cancer.
•
Iressa® is a tyrosine kinase inhibitor that may block the action of the over-expressed
EGF receptors on some lung cancer cells.
Enzyme-coupled signalling case study
JAK-STAT pathways receptors
•
This is another class of enzyme-coupled signal receptors.
•
They are receptors that always exist as two identical single-pass transmembrane proteins
that exist as homo dimers. Each of their cytoplasmic tails is bound to a janus kinase (JAK) .
•
Tyrosine residues are phosphorylated on the cytoplasmic domains on ligand binding, as
are associated signal transducer activator of transcription (STAT) molecules.
•
These STAT molecules dissociate to form cytoplasmic dimers, which enter the nucleus and
bind to specific DNA sequences in the promoters of genes that begin transcription.
•
They differ from RTKs in the directness of their response as few intermediary messengers
are utilised.
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Growth hormone, erythropoietin and interleukins act by these pathways.
Enzyme-coupled signalling case study
JAK-STAT pathways of growth hormone (STH)