Gprotein linked receptors and the second messenger cAMP

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G-protein linked receptorsand the second
messenger cAMP
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G-protein-linked receptors

• Largest family of cell-surface receptors and are
  found in all Eucaryotes
• Mediate the cellular response to an enormous
  diversity of signaling molecules, including
  hormones, neurotransmitters, odorants and
  photons.
• Adrenaline activates 9 distinct G-protein-coupled
  receptors, serotonin activates at least 15.
• Half of all known drugs work through G-protein
  linked receptors.

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Two major pathways by which G-protein linked
receptors signal.
Signaling molecule

• Most G-protein-linked receptors signal through
  the second messengers cyclic AMP or calcium.
• In both cases, the activated receptor binds to a
  trimeric G-protein to begin the signaling event.

receptor
G-protein
enzyme
cAMP
Ca2
Target protein
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G-protein-linked receptors functionally diverse
but share a common structure
Ligand binding
extra-cellular
Serpentine
single polypeptide chain -characterized by seven
hydrophobic stretches of 20-25 amino acids -
predicted to form transmembrane alpha helices
connected by alternating extracellular and
intracellular loops
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G-protein-linked receptors function through
trimeric G-proteins

• G-protein-linked receptors
• mediate their intracellular actions
• through target ion channels or enzymes.
• pathway always involves activation of one or
  more
• guanine nucleotide-binding regulatory proteins
  (trimeric G proteins).

• trimeric G proteins consist of three protein
  subunits alpha, beta and gamma.
• The G alpha binds a guanyl nucleotide.
• Various types, each specific for a set of
  serpentine receptors and for a particular
  downstream target, but they all operate the same
  way.

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The trimeric GTP-binding proteins act as
molecular switches

• Functionally couple the G-protein-linked
  receptors to their target enzymes
• or ion channels.
• GTPases that function as molecular switches
• flip between two states active and inactive.
• Inactive trimer bound to GDP through Ga
• ??????? Ga bound to GTP
• A G-protein which acts to stimulate a target
  enzyme is called a G stimulatory (Gs).
• Gi is inhibitory.

two conformation states
active
inactive
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Alpha subunit
Gamma subunit
Beta subunit
Binding of GTP induces large conformational
changes in the switch regions leading to
dissociation of the subunits.
The structure of a trimeric G-protein bound to
GDP based on x-ray crystallographic analysis
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Trimeric G-proteins functionally couple the
receptor to adenylyl cyclase to regulate the
production of cAMP
Adenylyl cyclase is the integral membrane
protein that catalyzes the cyclization of ATP.
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Adenylyl cyclase anchored by 2 sets of 6
transmembrane helices with active site made from
2 intracellular domains
outside
Lipid bilayer
cytosol
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Receptor, inactive G-proteins, and adenylyl
cyclase are within shouting distance in the
cell membrane.
Ligand binding (1) -conformational change in
the receptor -separates TMs. -separation of the
TMs may open a crevice for binding to
G. Receptor binds to G protein (2)
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• Receptor activation results in
• activation of adenylyl cyclase.
• -indirect
• -stimulates a trimeric G-protein
• -trimeric G-proteins dissasemble
• when activated.

Receptor binds to G-protein induces
conformational change (3) GDP is replaced by
GTP Ga dissociates from Gbg
The binding site for adenylyl cyclase is
unmasked.
Ga then binds to adenylyl cyclase
(4), activating synthesis of cAMP
A single hormone/ receptor complex stimulates the
production of many molecules of Gsa
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The binding of the Gas subunit to adenylyl
cyclase activates the enzyme to produce many
molecules of cAMP.
signal amplification
Binding of Ga to adenylyl cyclase causes a
conformation change in Ga and GTP is hydrolyzed
to GDP. This causes Ga to dissociate
from adenylyl cyclase and re-bind Gbg
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Terminating the response

• The hormone/receptor complex must be deactivated
  to return to
• the unstimulated state.
• -phosphorylation events on the carboxy terminal
  tail of the receptor
• lead to the inactivation of the receptor.
• Hydrolysis of GTP leads to inactivation of the
  trimeric G-protein.
• -enhanced by RGS proteins (regulator of G-protein
  signaling).

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Mammalian RGS proteins activate the GTPase
activities of G-protein alpha subunits
RGS proteins are GAPs (GTPase activating
proteins). -no effect on the time course of
nucleotide binding -but they stimulate the rate
of GTP hydrolysis.
GTP
GDP
a
GTP hydrolysis
a
b/g
g
RGS protein
b
MODEL RGS proteins accelerate GTP hydrolysis by
preferentially binding to and stabilizing G
proteins in their transition state for the
hydrolysis reaction.
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Inhibitory G proteins

• While the beta-adrenergic
• receptors are functionally
• coupled to G-stimulatory
• proteins, the alpha-2
• adrenergic receptors are
• coupled to inhibitory G
• proteins.

• G i can contain the same beta/gamma subunits as
  Gs, but the alpha subunits are different.
• G i inhibits adenylyl cyclase in an indirect
  manner.

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Hormone-induced activation and inhibition of
adenylyl cyclase is mediated by G-sa and G-ia
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G-protein linked receptors at a glance
Receptor ligand
Conformational change
Stimulates the G-protein to exchange bound GDP
for GTP
kinase
GDP
GTP
Ga stimulates AC to make cAMP
RGS
GTP
Adenylyl cyclase
cAMP
Second messenger
Each step amplifies the signal
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The second messenger Cyclic AMP
Adenine ring
Cyclic phosphate
Ribose sugar
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Cyclic AMP as a Second Messenger

• Cyclic AMP serves as an intracellular second
  messenger of many hormones neurotransmitters.

• Cyclic AMP is detected inside cells by protein
  kinase A (PKA) which then phosphorylates target
  proteins.

Terminating the response

• The only means of degrading cAMP is through the
  action of cAMP phosphodiesterases

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The activation of PKA

• PKA
• Serine/threonine kinase
• Accounts for the effects
• of cAMP in most cell types
• Two types
• -type I cytosol
• -type II anchored to membranes

inactive
PKA
inactive

• cAMP
• Binds to regulatory subunits of PKA
• induces a conformational
• change
• Regulatory subunits
• dissociate from catalytic
• subunits.

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Some Hormone-induced Cellular Responses Mediated
by cAMP
Triglyceride breakdown in fat mediated by
adrenaline. Increase in heart rate and force of
contraction in heart mediated by
adrenaline. Glycogen breakdown in muscle
mediated by adrenaline Changes in gene
transcription
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Regulation of glycogen breakdown and synthesis by
cAMP in liver and muscle
active PKA
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How does a rise in cAMP levels lead to altered
gene transcription?

• Increase in cAMP activates PKA
• PKA catalytic subunits move into
• the nucleus
• PKA phosphorylates the transcription
• factor CREB (cAMP response
• element binding protein).
• Phosphorylated CREB binds to the co-
• activator CBP
• Transcription is activated.

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Vibrio cholerae
Spread via -contaminated water -raw or
undercooked shellfish
problem in -developing nations -natural
disasters Symptoms -abrupt, painless, watery
diarrhea -metabolic acidosis with
potassium depletion -death
causative agent of cholera
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Cholera Toxin

• cholera toxin
• enzyme that catalyzes the transfer of ADP ribose
  from intracellular NAD to alpha s.
• The ADP ribosylation alters the alpha s so that
  it can no longer hydrolyze its bound GTP. Thus,
  alpha s continues to stimulate adenylyl cyclase
  to produce cAMP.
• The prolonged production of cAMP in the
  intestinal epithelial cells causes a large efflux
  of Na and water into the gut, and is responsible
  for the severe diarrhea that is characteristic of
  cholera.

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Effect of cholera toxin
Persistent activation of adenylyl cyclase
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Summary

• Gprotein-linked receptors
• Mediate the cellular response to an enormous
  diversity of signaling
• molecules, including hormones, neurotransmitters,
  odorants and photons
• Signal through trimeric G-proteins
• Trimeric G-proteins functionally couple their
  receptors to target enzymes
• Some trimeric G-proteins activate adenylyl
  cyclase to synthesize cAMP
• cAMP is an important second messenger that
  functions through
• PKA to regulate metabolism and transcription.