Transcript Control of Cell Function by 2nd Messenger Systems

2nd Messenger Systems,
continued
Cyclic AMP production and degradation
• In resting cells, the cAMP level is so low (10-8M) that it does
not bind the targets, such as regulatory subunits of cAMPgated channels. Stimulation of a G Protein Receptor raises
the level 100x, enough to saturate the receptors.
What if a G-Protein-adenylcyclase
system got stuck “on”?
• The cholera toxin is released in the gut by the bacteria
Vibrio cholerae. The toxin enzymatically alters Gαs so
that it no longer hydrolyzes GTP. The continuous
presence of stimulatory Gαs causes the intestinal cells
to secrete large amounts of salt and water, causing
diarrhea and therefore dehydration.
• The Bordetella pertussus toxin acts in a similar way on
the inhibitory G protein, but it is not apparent why it
causes whooping cough.
Cyclic AMP modulation of Protein Kinases
Effects of Protein Phosphorylation
Addition of a phosphate by protein kinase or removal
by protein phosphatase is the most common posttranslational modification of proteins. It turns
processes on and off, e.g.,
1. Cell motility
2. Membrane channels
3. Cell division
>99% of phosphorylation occurs on serine or threonine
residues.
The effects on structure include:
1. Steric interference: altering affinity
2. Conformational change that blocks or activates
enzymes
3. Creation of binding sites
Regulation of
protein kinase A
• The inactive form
consists of two
regulatory and two
catalytic subunits.
Binding of cAMP to the
regulatory subunits
induces a
conformational change
that allows the
enzymatically active
regulatory subunits to
dissociate.
Receptor Tyrosine Kinases
Receptors that are catalytic
The human genome encodes 59 receptors of this type –
most are for growth factors. The name receptor tyrosine
kinase refers to the fact that the intracellular domain of
these proteins has intrinsic kinase activity.
These receptors are unique in that the receptor is a dimer.
In some cases, interaction of the 1st message with the
extracellular domains of two receptor molecules causes
formation of the dimer. The intracellular domains then
phosphorylate each other. This activates the receptor,
which can proceed to phosphorylate particular tyrosines of
other proteins. In other cases (the insulin receptor), the
receptor is already a dimer and insulin binding simply
induces a conformational change.
Structures of receptor protein tyrosine kinases (FYI:
PDGF is platelet derived growth factor, EGF is epidermal growth factor)
Response sequence
• 1. Ligand-induced receptor dimerization
• 2. Autophosphorylation: polypeptide strands crossphosphorylate one another.
Response sequence, con’t.
• 3. This increases protein kinase activity AND
• 4. Phosphorylation of tyrosine residues creates binding sites for additional
proteins that transmit signals downstream. (SH2 is the region that binds)
• 5. The activation of the downstream signaling molecule is the first step in the
growth factor responses.
Link between a Receptor Protein Tyrosine Kinase
and a second-messenger system: Phospholipase C
• The SH2 domain of Phospholipase C-γ allows it to associate
with PTK and be localized near the membrane, where it can
attack a specific kind of membrane lipid, turning it into 2
signaling molecules.
• (The same reaction is generated by G-Protein activation of
Phospholipase C-β.)
• The target of Phospholipase C, Phosphotidylinositol 4, 5 bis
phosphate, is a minor membrane component, mainly found on
the inner half of the bilayer. Phospholipase C catalyzes
phosphotidylinositol 4,5-bisphosphate (PIP2) conversion to the
second messengers inositol trisphosphate (IP3) and the
corrresponding diacylglycerol (DAG). This reaction requires
phosphate donation by ATP.
Inositol Triphosphate (IP3) and Diacylglycerol
(DAG) as second messengers
Ligand-triggered sequence
One effect of IP3: release of Ca++ from the
endoplasmic reticulum
Control of Cell Function by 2nd
Messenger Systems
An example: Glycogen
metabolism
A push-pull hormonal system regulates plasma
glucose levels
• Glucagon and
epinephrine are
released when
plasma glucose levels
fall below about 5 mM
• Insulin is released
when plasma glucose
levels rise above
about 5 mM, and in
response to gut
signals that indicate
ingestion of a
carbohydrate meal
2 tissues, 3 hormones
Receptors/2nd
messengers
tissue
Hormones
Liver
Glucagon (from
Glucagon
pancreatic alpha cells) receptor/Gs G
protein/cAMP
degradation
β/ receptor/Gs G
protein/ cAMP
degradation
α1 Gq G protein/
IP3 /Ca++
degradation
Insulin (from
pancreatic beta cells)
Insulin
receptor/IRS
proteins
synthesis
Epinephrine
β receptor/Gs G
protein/ cAMP
degradation
Epinephrine (from
adrenal medulla)
Muscle
Net effects on
glycogen
Insulin has multiple intracellular
consequences
• The insulin receptor phosphorylates a
family of IRS (insulin receptor substrate)
proteins, which then activate other
downstream signaling proteins, leading to
a large variety of metabolic effects in the
target cells.
Major insulin effects that relate to
glycogen metabolism in liver
• Enhances activity of glycolytic enzymes (hexokinase,
phosphofructokinase, pyruvate kinase and pyruvate
dehydrogenase)
• Inhibits glucose-6-phosphatase
• Stimulates conversion of glycogen synthetase kinase
from active to inactive form
• Since glycogen synthetase kinase inactivates glycogen
synthetase, inactivating it stimulates glycogen
synthetase
• Inhibits glycogen phosphorylase
• (glucose uptake by liver is mainly via the insulininsensitive GLUT2 transporter)
Effect of insulin on liver
Glycogen
synthesis and
glycolysis are
stimulated;
gluconeogenesis
is inhibited
Major effects of insulin on glycogen
metabolism in muscle
Just as in liver, except that
• gluconeogenesis does not occur in muscle
• in muscle insulin stimulates insertion of
insulin-sensitive GLUT4 glucose
transporters into the plasma membrane
Effect of insulin on muscle
Glucose uptake,
glycolysis and
glycogen
synthesis are
stimulated –
gluconeogenesis
is not an issue in
muscle
Other metabolic effects of insulin
• Stimulates translation of mRNA into
protein
• Inhibits proteolysis
• Stimulates enzymes involved in
triglyceride synthesis and inhibits lipolysis
• Stimulates expression of genes involved in
tissue growth
Major effects of glucagon on
glycogen metabolism in liver
Glucagon antagonizes the effects of insulin
Promotes net glycogen breakdown: inhibits
hexokinase and glycogen synthetase and
activates glycogen phosphorylase and glucose6-phosphatase
Promotes gluconeogenesis by stimulating key
enzymes in the pathway – particularly ones
related to fructose phosphates, as shown in
following slides:
Glucagon and epi stimulate glycogen mobilization
by activating cAMP-dependent protein kinase A
Phosphorylase kinase b
Phosphorylase kinase a
Glycogen breakdown
activity increases
cAMP
Protein Kinase A
Glycogen synthetase a
Glycogen synthetase b
Glycogen synthesis
activity decreases
Active forms in black; inactive forms in red