Download BS2550 Lecture Notes cAMP

BS2550
These notes are intended only as a supplementary lecture summary and should be used in conjunction with other
lecture handouts and text and diagrams from textbooks (e.g. Lodish 4th Edn. Molecular Biology of the Cell Chapter
20, or Lodish Molecular Cell Biology 5th Edn.Chapt 13 Signalling at the Cell Surface). If you detect any errors or
require further clarification please inform Dr Davies The notesfor this course are also available on the School
Web-site – Click on For Students, then Undergraduate Teaching materials then BS2550.
HORMONES and SECOND MESSENGERS
A hormone is a molecule which is released from and endocrine cell in response to a
physiological signal, into the blood circulation and then affects the metabolism in one or more
target cells. Hormones are of two types (1) Those which can cross the plasma membrane, and
are taken up by the nuclei by binding to an intracellular receptor protein and generally have
specific effects on gene transcription (e.g. steroid and thyroid hormones) and (2) Those which
do not cross the plasma membrane but bind to a specific receptor protein on the outside of a
cell and cause changes in enzyme activity and metabolism, ion fluxes and/or gene
transcription within the cell. The second category can be subdivided as follows:
(a) hormones which cause opening/closing of ion-channels
(b) hormones which are coupled to a second messenger system via a G-protein
(c) hormones which bind to a transmembrane receptor which has a hormone binding site on
the outside of the membrane and a hormone activated enzyme catalytic site on the
cytoplasmic side. (e.g. Insulin Receptor Tyrosine Kinase)
Second Messenger
A second messenger is a low molecular weight metabolite released into the cell cytosol in
response to a hormone binding to a plasma membrane receptor. For a molecule to be
identified as s second messenger it must satisfy a number of criteria:
1) Changes in second messenger must reflect changes in external hormone concentration.
2) The second messenger must affect cell metabolism in a way consistent with the known
physiological effects of the hormone.
3) There must be a mechanism for the rapid removal of the second messenger thus
terminating the signal quickly, normally an enzymic mechanism
4) There should be a timed sequence:
change in [hormone]
-- [2nd messenger]
(+ve or -ve)
- [enzyme activity]
or [protein level]
(+ve or -ve)
and a causal relationship must be demonstrated between these changes.
5) The mechanism for the transmission of information must be elucidated i.e. SIGNAL
TRANSDUCTION MECHANISM. Each of the protein components of signal transduction
pathway and the nature of the interactions should be identified
6) Second messengers are not normally found on major metabolic pathways and are usually
present in cells at very low concentration but change rapidly by an order of magnitude or
more in response to hormonal stimulation.
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Examples of second messengers
cAMP
cGMP
(Ca2+)
Inositol phosphates e. g. IP3
Diacylglycerol (DAG)
Ceramide (N-Acyl sphingosine)
Phosphatidyl inositol phosphates {e.g. PtdIns(3,4,5)P3}
Phospho-oligosaccharides
Nitric Oxide
As is evident from this list, second messengers have a varied chemical nature and can be
hydrophilic and found in the cytosol or lipophilic and associated with membranes or in the
case of nitric oxide a gas which is non-polar can diffuse from cell to cell locally.
Glucagon and cAMP
Glucagon is a pancreatic hormone secreted in response to hypoglycaemia and is a key
regulator of hepatic glycogen breakdown and gluconeogenesis. The -cells of the Islets of
Langerhans produce a linear peptide of 29 amino acids. This hormone is very important in the
development of diabetes, in that it continues to be produced despite a lack of insulin,
contributing to the hyperglycemia found in this disease. The half-life of glucagon is very short
(~6min), it is taken up by the liver and kidney and inactivated. Glucagon is a powerful
hyperglycaemic agent which stimulates hepatic glycogen phosphorylase and gluconeogenesis
while simultaneously inhibiting glycogen synthase and glycolysis. Glucagon is thought to
exert these effects via changing the level of cAMP within the cell. (>structure of cAMP<)
The effects of cAMP are, in turn, mediated by cAMP-dependent protein kinase (PKA) which
phosphorylates specific Thr/Ser residues of key regulatory enzymes leading to either the
activation or inactivation of these enzymes. (note that glucagon is not the only hormone
which uses cAMP as a second messenger – other hormones in other tissues also regulate
cAMP with different consequences)
Glucagon Receptor (GR)
The receptor belongs to family of related hormone receptors which have 7 transmembrane
domains, with the hormone binding site on the outside of the cell, and with the receptor
coupled to G-proteins. The levels of this protein in the liver are down-regulated by cAMP and
up-regulated by glucose.
Adenylate Cyclase(AC)
The enzyme which synthesises cAMP is also a transmembrane protein with the active site on
the cytosolic face of the plasma membrane. It can be assayed by a radioactive method
involving radiolabeled [-32P]ATP as a substrate and monitoring the appearance of
[32P]cAMP. The substrate and the product are readily separated by an ion-exchange
chromatography by virtue of their different charges.
Evidence for the localisation of these proteins came from whole cell studies with adipocytes,
where the partial digestion of the outer plasma membrane with trypsin (trypsinisation)
results in loss of glucagon binding while the basal adenylate cyclase activity remained. In any
purification scheme involving the subcellular fractionation of cell membranes, AC, GR and
K+Na+ ATPase activity can be shown to co-localise, indicating the plasma membrane location
of these proteins.
Mobility of receptors
In avian erythrocytes AC is coupled to a -adrenergic receptor and therefore these cells
respond to catecholamine hormones, such as adrenaline with an increase in cAMP. The AC
activity can be destroyed by treating these cells with N-ethyl-maleimide while the receptor is
not affected. These cells therefore lose their ability to respond to adrenaline. You can fuse
these cells with another cell type which lacks receptor but has AC activity and the hybrid
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cell’s ability to respond to adrenaline is restored. Alternatively, liposomes containing adrenergic receptors can be fused with cells to make the cells responsive to adrenaline (see
Lodish Chapt 20). These experiments indicate that the receptor and the AC are free to move
and to interact within the plasma membrane.
In vitro experiments showing hormone sensitivity of liver plasma membrane
It is possible to isolate plasma membrane fragments which are capable of binding glucagon
and have AC activity. Glucagon binding can be assessed by incubating [125I]glucagon with
plasma membrane preparations, layering the mixture on to 20% sucrose in a centrifuge tube
and the centrifuging the plasma membrane through the sucrose, the unbound hormone
remains at the interface whilst the bound hormone sediments to the bottom of the tube. A
kinetic analysis indicates that this binding is very tight (KD = 3nM) and very specific in that
even closely related peptide hormones fail to either prevent or displace the glucagon binding.
Initially this binding and the subsequent activation of AC, was thought to be rather slow
compared to what happens in the intact cell until it was realised that GTP (at concentrations
0.1 10 ) was also required in the in vitro system. GTP is not a substrate for AC but
rather appeared to be acting as an allosteric activator of the system, together with glucagon.
Half-maximal activation of AC occurs with ~4 nM glucagon corresponding to the KD
described above. GTP increases the affinity of the membrane preparation for glucagon as well
as accelerating the effect of the hormone.
Discovery of G-proteins
The function of GTP in the signal transduction system was not understood until the discovery
of the G-proteins. There is a lymphoma cell line mutant called S49 (cyc-) which apparently
lacked AC because it did not respond in the same way to adrenaline (an increase in cAMP) as
the wild type cell, despite the presence of a normal adrenaline receptor. It was expected that if
you treat plasma membrane from (cyc-) cells with an extract from wild type cells you can
restore the hormone sensitivity and sure enough you can! However, the importance of control
experiments in Biochemistry is evident in a further study in which the AC in the extract was
inactivated by heating or NEM treatment; when these extracts are added to the (cyc-)
membranes, you also get restoration of hormone sensitivity! The explanation for this apparent
discrepancy is not that it is AC which is missing in the (cyc-) cells but another protein
component, which was subsequently found to be a G-protein. The (cyc-) membrane proved to
be useful for the assay of G-protein activity.
Assay of G-proteins
G-proteins may be assayed by a variety of methods including:
(1) activation of AC in (cyc-) plasma membrane by extracts containing G-protein
 ability of the G-protein to bind GTP and GDP analogues e.g. GDP--S
 the intrinsic GTPase activity of G-proteins can be measured
Properties of Mammalian Heterotrimeric G-proteins
The heterotrimeric G-proteins are Guanylyl nucleotide-binding proteins which are members
of a very versatile family of proteins which sort and amplify all sorts of different signals in
different tissues. These membrane proteins have been shown to interact with many different
hormone signalling systems which can either activate (Gs proteins) or inhibit (Gi proteins)
adenylate cyclase in different signal transduction mechanisms. Another type of G-protein Gq
is involved in activating phospholipase C, as we shall see later. G-proteins are located at the
cytoplasmic face of the plasma membrane anchored to the membrane by a lipid anchor. They
are heterotrimers made up of an -subunit of 40-45 KDa, a -subnit of ~35 KDa and a -subunit
of 7 KDa.The -subunits are very variable between signal transduction systems, while there is
less variation in the - and -subunits but multiple forms of these have been reported. The
hormone-receptor complex does not directly activate AC but rather it activates the G-proteins
which interacts with AC and either activates or inhibits AC activity.
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Sequence of events in the function of G-protein







The  complex is the inactive form of the G-protein which has GDP bound
The hormone binds to the receptor to form a HR complex but each interaction is very
short-lived (<1 sec)
The active HR complex allows GDP to dissociate from the -subunit and GTP then
associates with it.
The subunit dissociates from the -subunits
The subunit binds and activates AC and cAMP is generated
The subunit has an intrinsic GTPase activity which converts the GTP to GDP allowing
for the inactivation of the subunit by reassociation with the -subunits.
The subunit remains active for say 10 sec before the GTP is converted to GDP
The G-protein is acting as a ‘switch’ and a ‘timer’ which translates the hormone binding
event into a membrane signal which activates AC and the signal remains active for some time
after the hormone has dissociated. This is characteristic of hormonal signalling events which
can have consequences long after the hormonal changes have returned to basal levels.

The -subunits have another function in some systems eg in brain Type 1 AC 
stimulates while -subunits inhibit.

Specificity of Signal Transduction
Each hormone interacts with a specific receptor with high affinity, each HR complex interacts
with a specific G-protein and each subunit interacts with its correct effect protein e.g. AC
or phospholipase. This explains how different hormones can use similar components to
specifically regulate particular functions if diverse target cells.
Investigation of G-protein Function
GTP and GDP analogues
Nucleotide analogues (>structures<) are useful tools in the study of G-proteins
e.g. Guanylyl imidodiphosphate (GppNp), Guanylyl methylene diphosphate (GppCH2p) or
GTPS can all bind to and activate Gs proteins and furthermore, they keep the protein in the
dissociated, activated state because they are not readily hydrolysed by the GTPase activity.
Conversely GDP analogues such as GDPS bind to and maintain the G-protein in the inactive
state.
Cholera Toxin (CX)
This is another useful tool in the study of G-proteins. The toxin is an enzymatic protein,
produced by the microorganism Vibrio Cholerae, which is capable of being taken up by
mammalian cells. The protein has a transferase activity which transfers ADP-ribose moiety
from NAD+ to a specific arginine residue in the -subunit. This has the effect of persistently
inhibiting GTPase activity and thus the Gs-protein remains in a permanently activated state,
which in turn activates AC and raises cAMP levels in the cell. (in Cholera the toxin is taken
up by cells lining the intestine, and promotes an increase in cAMP resulting in an
uncontrolled secretion of Na+ , HCO3- and water giving rise to the characteristic symptoms of
salt-loss and dehydration characteristic of Cholera)
Pertussis Toxin (PX)
This is a similar toxin from Bordetella pertussis (the organism which causes whooping
cough) which affects the lining of the respiratory tract, In this case it is a Cys residue in a Gi
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protein which is ADP-ribosylated. This prevents GTP/GDP exchange and thus prevents
inhibition of AC by Gi. (In this condition there is a massive secretion of fluid and mucous
which results in paroxysmal fits of coughing with a characteristic whooping sound). Again
this an useful tool for the study of Gi proteins and their function.
Regulation of cAMP levels in Cells
Adenylate Cyclase (AC)
This is the low abundance, plasma membrane associated enzyme, with an active site on the
cytoplasmic face of the membrane which catalyses the conversion of ATP to cAMP and PPi.
It is a large multigene family which may be activated by Gs or inhibited by Gi. AC consists of
12 trans-membrane domains and two conserved cytosolic loops (between TM 6-7 and the Cterminal sequence) which form the active site. The enzyme can be activated by forskolin, a
plant terpenoid, which is cell-permeable and an useful tool for the study of the function of AC
in mammalian cells because it can increase cAMP in cells in the absence of hormones such as
glucagon or adrenaline.
The G-protein is thought to bind specifically to TM6-7 in order to activate or inactivate AC.
There are also phosphorylation and calmodulin binding sites which regulate the degree of
activation of AC which allows cross-talk (transfer of information) between cAMP and Ca2+
signal transduction mechanisms.
cAMP Phosphodiesterase (PDE)
PDEs play an important role in the termination of the hormonal cAMP transduction systems
by the rapid removal of the second messenger. It can be assayed using [3H]cAMP as substrate
and converting the product 5’AMP into [3H] adenosine with an excess of 5’nucleotidase. The
tritiated product is readily separated from the substrate using ion-exchange matrix.
There are a number of different PDEs with different kinetic and regulatory properties and
with differing cellular locations. Some PDEs bind calmodulin and are therefore regulated by
the intracellular free Ca2+, others by phosphorylation by PKA and by insulin-generated
signals.
The Regulation of [cAMP] by the coordinated control of AC and PDE
The basal level of cAMP in most mammalian cells is ~ 1 M and about 10 M is required to
activate PKA. This threshold can be reached by activating AC, by inhibiting PDE or by a
combination of the two effects.
In some cell types (e.g. adipocytes) both activities are high with a potential for ‘futile’ cycling
(in effect the wasteful hydrolysis of ATP by the rapid synthesis and degradation of cAMP
occurring simultaneously). This allows the cAMP levels to be regulated by small changes in
the activity of either AC or PDE or both. In other cell types such as the hepatocyte both
activities are relatively low and [cAMP] is raised by activating AC but the duration of the
signal is regulated by PDE.
Compartmentation of cAMP, AC and PDE
The second messenger cAMP is found in the cytoplasmic compartment of the cell. It is
generated at the plasma membrane but degraded either by PDE in the cytoplasm or by PDE
anchored to an intracellular membrane. The consequence is that there is a gradient of [cAMP]
concentration from the plasma membrane to the intracellular organelles. Similarly the
location of PKA may be free in the cytosol or may be anchored via its Regulatory subunit to
some intra-cellular location. The effectiveness of the second messenger can therefore depend
on the location of the cAMP as well as its concentration
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Drugs which inhibit PDE
The xanthine analogues caffeine, theophylline (found in tea and coffee) and the synthetic
IBMX (isobutyl methyl xanthine) (>structures<) are often use in conjunction with a
hormone, cholera toxin or forskolin to raise intracellular cAMP and to prolong the signal.
These xanthine derivatives are inhibitors of PDE and produce their well-known effects in
humans by raising cAMP in various tissues.
How do transient changes in cAMP regulate metabolism?
When a hormone binding event occurs the signal transduction mechanism is initiated and
there is a rapid rise in cAMP within the matter of seconds. This rise is transitory and the level
of cAMP decreases to basal levels within minutes. This gives rise to a similar change in PKA
activity. Protein kinase A (PKA) is an enzyme which catalyses the phosphorylation of a
number of other proteins at specific serine or threonine residues. These proteins may be key
regulatory enzymes in metabolism (e.g pyruvate kinase) or other protein kinases ( e.g.
phosphorylase kinase) which can become activated and themselves phosphorylate other
proteins. These covalent modifications (also known as a stable change because the covalent
modification of the protein will survive dialysis or gel filtration which is not the case when an
enzyme is activated or inhibited by an allosteric effector molecule) result in a co-ordinated
series of metabolic events which allows the tissue to respond in an appropriate fashion to the
hormonal signal.
One example is found in action of glucagon in the liver, the hormone increases [cAMP],
which activates PKA, which, in turn, phosphorylates and activates phosphorylase kinase,
which in turn, phosphorylates and activates phosphorylase the enzyme which breaks down
glycogen and increases the blood glucose. At each stage the signal is amplified and prolonged
in that each successive event in the signalling pathway lasts for a longer time and the effect
continues long after the hormone has become dissociated from its receptor
Mechanism of PKA activation
ALL the known effects of cAMP in mammalian cells are mediated by the activation of PKA
by the following mechanism:
C2R2
 2C + 2R
The inactive enzyme is the C2R2 form, which is a complex of two regulatory and two catalytic
subunits. The increase in cAMP results in the binding of the second messenger to the R
subunits which, in turn, causes the complex to dissociate and the C subunit is released and is
therefore activated. When the cAMP levels fall the cAMP dissociates from R and this allows
the inactive C2R2 complex to be re-formed.
This can be shown experimentally with partially purified PKA. If you add [3H]cAMP to PKA
and apply to a gel filtration column, the larger molecular weight cAMP- binding subunit
separates from the catalytic subunit (you can show this by measuring cAMP binding and
protein kinase activity). If you dialyse off the cAMP from R and recombine with C then the
protein kinase activity is inhibited because R + C form a complex.
Assay of PKA
In order to measure the activity of a protein kinase you need to incubate the enzyme with the
protein substrate (or a peptide with a sequence similar to the protein) with [32P]ATP. This
means that the terminal phosphate on the ATP is labelled and it is this which is transferred to
a serine or threonine hydroxyl group and as a result the protein (or peptide) becomes labelled.
The protein can then be precipitated with TCA to remove the unreacted ATP or alternatively
separated from ATP by ion-exchange chromatography or immunoprecipitation.
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What happens when a protein is phosphorylated?
The phosphorylation results in a conformational change in the protein and as a result the
kinetic properties of the protein/enzyme are changed – the protein may become activated or
inactivated. Protein kinase activity can also therefore be assayed by measuring some aspect of
the kinetics if the target protein Some examples of PKA substrates are: tyrosine hydroxylase,
phosphorylase kinase (and-subunits), PFK2/FBPase2, glycogen synthase, pyruvate
kinase, CREB and a number of others.
You can show that a range of proteins become phosphorylated if you preincubate hepatocytes
with 32PO4 (this is incorporated into cellular ATP) and then add glucagon, wait a few minutes
and then extract the protein from the cells and subject the protein to SDS/PAGE
electrophoresis and then subject the gel to autoradiography. By comparing the autorad. to that
from extracts of cells not incubated with glucagon you can identify at least 12 bands whose
phosphorylation is increased by the hormone. Some of these can be easily identified from
their M.Wt. as subunits of phosphorylase kinase, phosphorylase, glycogen synthase, pyruvate
kinase etc.
Effect of phosphorylation on enzyme kinetics (e.g. Glucose metabolism)
When phosphorylase kinase is phosphorylated by PKA, its Vmax is increased leading to an
increase in phosphorylase Vmax and the breakdown of glycogen occurs. When glycogen
synthase is phosphorylated the Vmax decreases and glycogen synthesis ceases. This avoids
the energetically wasteful simultaneous synthesis and degradation of glycogen.
In many cases the Vmax is altered by covalent modification but there are also cases where the
Km of an enzyme is changed. For example, pyruvate kinase is a key regulator of hepatic
glycolysis (and therefore of gluconeogenesis). This is an allosteric enzyme which operates in
the cell at rates well below its potential Vmax. It is controlled by allosteric effectors such as
Fru -1,6P2 (an activator) and ATP and alanine (inhibitors). These increase the activity of the
enzyme by a decrease in the S0.5 (or apparent Km – note that allosteric enzymes do not obey
Michelis-Menten kinetics). These changes are not stable in that when the effectors are
removed the enzyme activity returns to the basal state. But when the enzyme is
phosphorylated by PKA the S0.5 is increased leading to an inhibition of the activity in vivo. In
this state the modification in kinetic properties will survive purification, hence this is a stable
change. The enzyme will only return to its basal activity when it is enzymatically
dephosphorylated by a Protein Phosphatase (see below).
What determines if a protein is phosphorylated?
The accessibility of the Ser/Thr residue is important as well as the sequence of amino acids
near the target site. A PKA phosphorylation motif usually consists of two basic amino acids
separated by an unspecified amino acid from the serine or threonine residue to be
phosphorylated. (e.g -X-R-R-X-S-X- ). Furthermore, the site needs to be at or near the surface
of the protein ,therefore the conformation of the protein is important. Other protein kinases
target different Ser or Thr residues and a different class of protein kinases, the tyrosine
kinases target Tyr residues.
There are many hundreds of protein kinases in mammalian cells each with its own function
and similarly there are hundreds of proteins which are regulated by reversible
phosphorylation/dephosphorylation. In these lectures we are interested in the so-called AGC
sub-family of protein kinases, which includes PKA, PKB (see Dr Crompton’s lectures),
PDK1, PKC and PKG.
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Hormonally induced changes are normally reversible – how is the protein
dephosphorylated?
There are a number of classes of Protein Phosphatases which can dephosphorylate proteins
by cleaving the covalently linked phosphate group and therefore reversing the effects of the
protein kinase. They are often distinguished by using specific inhibitors, which are often
natural products produced by micro-organisms.
PP1 dephosphorylates a number of proteins including phosphorylase a, phosphorylase kinase
and glycogen synthase. PP1 is inhibited by okadaic acid at concentrations above 10nM and by
tautomycin.
PP2A dephosphorylates a number of proteins including Pyruvate Kinase (therefore increasing
its activity- see above), PFK2/FBPase2, Acetyl Co A carboxylase. PP2A is usually found
associated with a 66 KDa Regulatory sub-unit which regulates its activity and location in the
cell. It is strongly inhibited by a cell-permeable PP2A inhibitor, okadaic acid at a
concentration of 1nM which is often used to diagnose the presence of the enzyme.
PP2B is a Ca2+/calmodulin dependent protein phosphatase – the Ca2+ chelator, EGTA,
inhibits its activity
PP2C is a Mg2+-dependent phosphatase
Further classes of protein phosphatases, PP4, PP5 & PP6 have been found more recently but
relatively little is known about the function of these enzymes
More on Protein Phosphatase inhibitors
Okadaic acid is a polyether fatty acid, and is associated with diarrhoeic seafood poisoning. It
is produced by dinoflagellates, marine plankton which can concentrate in scallops, mussels
and clams and can produce catastrophic results if these seafoods are eaten out of season,
presumably because of the inhibition of protein phosphatase in the cells lining the intestine. It
is a useful tool in that it is cell permeable and specific to PP2A and to a lesser extent PP1.
Tautomycin inhibits PP1 more than PP2A
Microcystin inhibits both but cannot readily cross the cell membrane and therefore is very
useful for in vitro work but not in vivo studies with intact cells.
Calyculin and Cantharidin are also inhibitors used to study protein phosphatases.
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