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G Protein Linked Receptors
Introduction
• G-protein-linked receptors are the largest family of cell-surface receptors.
• More than 100 members have already been defined in mammals.
• G-protein-linked receptors mediate the cellular responses to an
enormous diversity of signaling molecules, including hormones,
neurotransmitters, and local mediators, which are as varied in structure
as they are in function: the list includes proteins and small peptides, as
well as amino acid and fatty acid derivatives.
• The same ligand can activate many different family members.
• At least 9 distinct G-protein-linked receptors are activated by adrenaline,
for example, another 5 or more by acetylcholine, and at least 15 by
serotonin.
• Despite the chemical and functional diversity of the signaling molecules
that bind to them, all of the G-Protein-linked, G-protein-linked receptors
whose amino acid sequences are known from DNA sequencing studies
have a similar structure and are almost certainly evolutionarily related.
• They consist of a single polypeptide chain that threads back and forth
across the lipid bilayer seven times
A schematic drawing of a G-proteinlinked receptor. Receptors that bind
protein ligands have a large
extracellular ligand-binding domain
formed by the part of the
polypeptidemchain shown in light
green. Receptors for small ligands
such as adrenaline have small
extracellular domains, and the ligandbinding site is usually deep within the
plane of the membrane, formed by
amino acids from several of the
transmembrane segments. The parts
of the intracellular domains that are
mainly responsible for binding to
trimeric G proteins are shown in
orange,while those that become
phosphorylated during receptor
esensitization are shown in red
Trimeric G Proteins Relay the Intracellular Signal from G
protein- linked Receptors
• The trimeric GTP-binding proteins (G proteins) that functionally couple
these receptors to their target enzymes or ion channels in the plasma
membrane are structurally distinct from the single chain GTP-binding
proteins (called monomeric GTP-binding proteins or monomeric GTPases)
that help relay intracellular signals and regulate vesicular traffic and many
other processes in eucaryotic cells.
• Both classes of GTP-binding proteins, however, are GTPases and function
as molecular switches that can flip between two states: active, when GTP
is bound, and inactive, when GDP is bound.
• When an extracellular ligand binds to a G-protein-linked receptor, the
receptor changes its conformation and switches on the trimeric G proteins
that associate with it by causing them to eject their GDP and replace it
with GTP.
• The switch is turned off when the G protein hydrolyzes its own bound GTP,
converting it back to GDP.
• But before that occurs, the active protein has an opportunity to diffuse
away from the receptor and deliver its message for a prolonged period to
its downstream target.
• Most G-protein-linked receptors activate a chain of
events that alters the concentration of one or more
small intracellular signaling molecules.
• These small molecules, often referred to as
intracellular mediators (also called intracellular
messengers or second messengers), in turn pass the
signal on by altering the behavior of selected cellular
proteins.
• Two of the most widely used intracellular mediators
are
• cyclic AMP (cAMP) and
• Ca2+: changes in their concentrations are stimulated
by distinct pathways in most animal cells, and most Gprotein-linked receptors regulate one or the other of
them
Two major pathways by which Gprotein-linked cell-surface receptors
generate small intracellular mediators.
In both cases the binding of an
extracellular ligand alters the
conformation of the cytoplasmic
domain of the receptor, causing it to
bind to a G protein that activates (or
inactivates) a plasma membrane
enzyme. In the cyclic AMP (cAMP)
pathway the enzyme directly produces
cyclic AMP. In the Ca2+ pathway the
enzyme produces a soluble mediator
that releases Ca2+ from the
endoplasmic reticulum. Like other
small intracellular mediators, both
cyclic AMP and Ca2+ relay the signal by
acting as allosteric effectors: they bind
to specific proteins in the cell, altering
their conformation and thereby their
activity.
Some Receptors Increase Intracellular Cyclic AMP by
Activating Adenylyl Cyclase via a Stimulatory G Protein
(Gs)
• Cyclic AMP was first identified as an intracellular mediator of
hormone action in 1959 and has since been found to act as an
intracellular signaling molecule in all procaryotic and animal cells
that have been studied.
• For cyclic AMP to function as an intracellular mediator, its
intracellular concentration (normally <10-7 M) must be able to
change up or down in response to extracellular signals: upon
hormonal stimulation, cyclic AMP levels can change fivefold in
seconds.
• Such responsiveness requires that rapid synthesis of the molecule
be balanced by rapid breakdown or removal.
• Cyclic AMP is synthesized from ATP by a plasma-membrane-bound
enzyme adenylyl cyclase, and it is rapidly and continuously
destroyed by one or more cyclic AMP phosphodiesterases, which
hydrolyze cyclic AMP to adenosine 5‘- monophosphate (5'-AMP)
Cyclic AMP. It is shown as a formula, a ball-and-stick model, and as a spacefilling model. (C, H, N, O, and P indicate carbon, hydrogen, nitrogen, oxygen,
and phosphorus atoms, respectively.)
The synthesis and degradation of cyclic AMP
(cAMP). A pyrophosphatase makes the
synthesis of cyclic AMP an irreversible reaction
by hydrolyzing the released pyrophosphate
• Many extracellular signaling molecules work by
controlling cyclic AMP levels, and they do so by altering
the activity of adenylyl cyclase rather than the activity
of phosphodiesterase.
• Just as the same steroid hormone produces different
effects in different targe cells, so different target cells
respond very differently to external signals that change
intracellular cyclic AMP levels.
• All ligands that activate adenylyl cyclase in a given type
of target cell, however, usually produce the same
effect:
• at least four hormones activate adenylyl cyclase in fat
cells, for example, and all of them stimulate the
breakdown of triglyceride (the storage form of fat) to
fatty acids
Adenylyl cyclase. In vertebrates the enzyme usually contains about 1100 amino
acid residues and is thought to have two clusters of six transmembrane segments
separating two similar cytoplasmic catalytic domains. There are at least six types
of this form of adenylyl cyclase in mammals (types I-VI). All of them are
stimulated by Gs, but type I, which is found mainly in the brain, is also stimulated
by complexes of Ca2+ bound to the Ca2+-binding protein calmodulin
• The different receptors for these hormones activate a
common pool of adenylyl cyclase molecules, to which
they are coupled by a trimeric G protein.
• Because this G protein is involved in enzyme activation,
it is called stimulatory G protein (Gs).
• Individuals who are genetically deficient in Gs show
decreased responses to certain hormones and,
consequently, have metabolic abnormalities, abnormal
bone development, and are mentally retarded.
• The best-studied examples of receptors coupled to the
activation of adenylyl cyclase are the b-adrenergic
receptors, which mediate some of the actions of
adrenaline and noradrenaline
Trimeric G Proteins Are Thought to Disassemble
When Activated
• A trimeric G protein is composed of three different polypeptide
chains, called a, b, and g.
• The Gs a chain(as) binds and hydrolyzes GTP and activates adenylyl
cyclase.
• The Gs b chain and g chain form a tight complex (bg), which anchors
Gs to the cytoplasmic face of the plasma membrane, at least partly
by a lipid chain (a prenyl group) that is covalently attached to the g
subunit.
• In its inactive form Gs exists as a trimer with GDP bound to as.
• When stimulated by binding to a ligand activated receptor, as
exchanges its GDP for GTP.
• This is thought to cause as to dissociate from b g, allowing as to
bind instead to an adenylyl cyclase molecule, which it activates to
produce cyclic AMP.
• If cells are to be able to respond rapidly to changes
in the concentration of an extracellular signaling
molecule, the activation of adenylyl cyclase must
be reversed quickly once the signaling ligand
dissociates from its receptor.
• This ability to respond rapidly to change is assured
because the lifetime of the active form of as is
short: the GTPase activity of as is stimulated when
as binds to adenylyl cyclase, so that the bound
GTP is hydrolyzed to GDP, rendering both as and
the adenylyl cyclase inactive.
• The as then reassociates with bg to re-form an
inactive Gs molecule
Assignment:
Role of G protein in Cholera
Some Receptors Decrease Cyclic AMP by Inhibiting Adenylyl Cyclase
via an Inhibitory Trimeric G Protein (Gi)
• The same signaling molecule can either increase or decrease the intracellular
concentration of cyclic AMP depending on the type of receptor to which it
binds.
• When adrenaline binds to b- adrenergic receptors, for example, it activates
adenylyl cyclase, whereas when it binds to a2- adrenergic receptors, it inhibits
the enzyme.
• The difference reflects the type of G proteins that couple these receptors to the
cyclase.
• While the b-adrenergic receptors are functionally coupled to adenylyl cyclase
by Gs, the a2-adrenergic receptors are coupled to this enzyme by an inhibitory
G protein (Gi).
• Gi can contain the same b-g complex as Gs, but it has a different a subunit (ai).
• When activated, a2-adrenergic receptors bind to Gi, causing ai to bind GTP and
dissociate from the bg complex.
• Both the released ai and bg are thought to contribute to the inhibition of
adenylyl cyclase.
• ai inhibits the cyclase, probably indirectly, whereas bg may inhibit cyclic AMP
synthesis in two ways - directly, by binding to the cyclase itself, and indirectly,
by binding to any free as subunits in the same cell, thereby preventing them
from activating cyclase molecules.
Assignment:
Role of G protein in Pertusis
Cyclic-AMP-dependent Protein Kinase (A-Kinase)
Mediates the Effects of Cyclic AMP
• Cyclic AMP exerts its effects in animal cells mainly by
activating the enzyme cyclic-AMP dependent protein kinase
(A-kinase), which catalyzes the transfer of the terminal
phosphate group from ATP to specific serines or threonines
of selected proteins.
• A-kinase is found in all animal cells and is thought to
account for all of the effects of cyclic AMP in most of these
cells.
• In the inactive state A-kinase consists of a complex of two
catalytic subunits and two regulatory subunits that bind
cyclic AMP.
• The binding of cyclic AMP alters the conformation of the
regulatory subunits, causing them to dissociate from the
complex.
• The released catalytic subunits are thereby activated to
phosphorylate specific substrate protein molecules
The activation of cyclic-AMP-dependent protein kinase (A-kinase). The binding of cyclic
AMP to the regulatory subunits induces a conformational change, causing these subunits
to dissociate from the complex, thereby activating the catalytic subunits. Each regulatory
subunit has two cyclic-AMP-binding sites, and the release of the catalytic subunits
requires the binding of more than two cyclic AMP molecules to the tetramer. This greatly
sharpens the response of the kinase to changes in cyclic AMP concentration. There are at
least two types of A kinase in most mammalian cells: type I is mainly in the cytosol,
whereas type II is bound via its regulatory subunit to the plasma membrane, nuclear
membrane, and microtubules. In both cases, however, once the catalytic subunits are
freed and active, they can migrate into the nucleus (where they can phosphorylate gene
regulatory proteins), while the regulatory subunits remain in the cytoplasm.
Glycogen metabolism in skeletal muscle cells
• Glycogen is the major storage form of glucose, and both its
synthesis and degradation in skeletal muscle cells are regulated by
adrenaline.
• When an animal is frightened or otherwise stressed, for example,
the adrenal gland secretes adrenaline into the blood, "alerting"
various tissues in the body.
• Among other effects, the circulating adrenaline induces muscle cells
to break down glycogen to glucose 1-phosphate and at the same
time to stop synthesizing glycogen.
• The glucose is then oxidized by glycolysis to provide ATP for
sustained muscle contraction.
• In this way adrenaline prepares the muscle cells for anticipated
strenuous activity.
• Adrenaline acts by binding to b-adrenergic receptors on the muscle
cell surface, thereby causing an increase in the level of cyclic AMP in
the cytosol.
• The cyclic AMP activates A-kinase, which phosphorylates two other
enzymes.
• The first, phosphorylase kinase, which was the first
protein kinase to be discovered (in 1956), phosphorylates in turn the enzyme glycogen phosphorylase,
thereby activating the phosphorylase to release
glucose residues from the glycogen molecule
• The second enzyme phosphorylated by activated Akinase is glycogen synthase, which performs the final
step in glycogen synthesis from glucose.
• This phosphorylation inhibits the enzyme's activity,
thereby shutting off glycogen synthesis.
• By means of this cascade of interactions, an increase in
cyclic AMP levels both stimulates glycogen breakdown
and inhibits glycogen synthesis, thus maximizing the
amount of glucose available to the cell.
The stimulation of glycogen breakdown by cyclic AMP in skeletal muscle cells. The
binding of cyclic AMP to A-kinase activates this enzyme to phosphorylate and thereby
activate phosphorylase kinase, which in turn phosphorylates and activates glycogen
phosphorylase, the enzyme that breaks down glycogen. The A-kinase also directly and
indirectly increases the phosphorylation of glycogen synthase, which inhibits the
enzyme, thereby shutting off glycogen synthesis.
• In some animal cells an increase in cyclic AMP activates the
transcription of specific genes.
• In cells that secrete the peptide hormone somatostatin, for
example, cyclic AMP turns on the gene that encodes this
hormone.
• The regulatory region of the somatostatin gene contains a
short DNA sequence, called the cyclic AMP response element
(CRE), that is also found in the regulatory region of other
genes that are activated by cyclic AMP.
• This sequence is recognized by a specific gene regulatory
protein called CRE-binding (CREB) protein.
• When CREB is phosphorylated by A kinase on a single serine
residue, it is activated to turn on the transcription of these
genes; the phosphorylation stimulates the transcriptional
activity of CREB without affecting its DNA-binding
properties.
• If this serine residue is mutated, CREB is inactivated and no
longer stimulates gene transcription in response to a rise in
cyclic AMP levels.