Transcript A. G protein–linked receptors

Cell Signaling
I. OVERVIEW
• Soluble chemical signals sent from one cell
to another are essential for communication
• The cellular recipient of the signal is known
as target cell and binds the signaling
molecule via a protein receptor (on cell
surface/cytoplasm or nucleus)
• Binding to receptor initiates signaling 
cascade of rxns that amplify signal and
produce effect.
G-protein Signaling
• G proteins are intracellular signaling
proteins that are named for their ability to
bind to GTP. They also possess GTPase
activity.
• Two categories of G proteins are described:
- heterotrimeric G proteins and
- the Ras super-family of G proteins
• Ras superfamily members a.k.a “small G
proteins” as they are monomers that
resemble one subunit of the heterotrimeric
G proteins.
• Ras proteins receive their signals from
catalytic receptors that have been activated
by their ligand.
• The overall effects of Ras signaling often
involve induction of cell proliferation, cell
differentiation, or vesicle transport.
Heterotrimeric and Ras superfamily G proteins.
• Heterotrimeric G proteins consist of three
subunits, α, β, and γ. The signaling process is
initiated by ligand binding to receptors linked to
G proteins tethered to the inner membrane leaflet.
• Activation of the G protein then enables it to
regulate a specific membrane-bound enzyme.
Products of reactions catalyzed by activated
enzymes include second messengers that amplify
the signal sent to the cell by the hormone or
neurotransmitter that bound to its receptor and
acted as the first message.
• Many second messengers activate
serine/threonine protein kinases, enzymes
that phosphorylate their substrates on
serine and threonine amino acid residues.
Changes in phosphorylation status of target
proteins, many of which are enzymes, can
alter their activity. The overall result is the
biological response of the cell to the
hormone or neurotransmitter. The
biological response is often the regulation
of a biochemical pathway or the expression
of a gene.
Overview of G protein signaling
I. RECEPTORS AND HETEROTRIMERIC G
PROTEIN SIGNALING
• Many hormones and neurotransmitters have
receptors on their target cells that are linked to
G proteins. G protein–linked receptors are the
most common form of cell surface receptor.
• These receptors have extra-cellular hormonebinding regions as well as intracellular
portions that interact with the G protein to
send the message from the hormone into the
cell to evoke a response.
A. G protein–linked receptors
• G protein–linked receptors are transmembrane
proteins with seven membrane-spanning
regions.
• Close to 400 distinct G protein–coupled
receptors have been identified in humans.
• Most are expressed in multiple tissues. Over
90% of them are expressed in the brain. All use
the same basic process to stimulate G proteins
to regulate the production of second
messengers.
Structure of G protein–coupled receptors.
B. Signaling mechanism
• All heterotrimeric G proteins use the same basic
scheme shown for the Gs type of G protein.
• An unoccupied G protein–linked receptor does not
interact with the G protein in close proximity to its
intracellular domain.
• Ligand binding to the receptor creates an occupied
receptor that undergoes a conformational change
and is then able to interact with the G protein.
• In response to the receptor binding to the G protein
complex, the Gα subunit of the G protein releases
GDP and binds GTP.
• The G protein is now active and the α subunit
dissociates from the β and γ subunits.
• The active α subunit then interacts with an enzyme
whose function is regulated by the G protein.
• Adenylyl cyclase is the enzyme activated by Gs
protein signaling to have the ability to convert ATP
to cyclic AMP (cAMP) and inorganic phosphate
(PPi).
• cAMP is the second messenger in Gs signaling. The
type of G protein that is activated and the second
messenger it regulates depend on the ligand, the
type of receptor, and the type of target cell.
• When hormone is no longer present, the receptor
will revert to its resting state. GTP is hydrolyzed to
GDP (by the GTPase of the G protein), the enzyme,
such as adenylyl cyclase, is inactivated, and the α
subunit will reassociate with β and γ subunits to
stop the signaling process.
III. HETEROTRIMERIC G PROTEINS AND THE
SECOND MESSENGERS THEY REGULATE
• Distinct members of the heterotrimeric G
protein family exist through the association of
various forms of the three subunits, α, β, and γ.
• At least 15 different α subunits are known.
Combinations of different α, β, and γ subunits
form the heterotrimeric subunits.
• GDP is bound to the α, subunit of the G
protein when all three subunits are joined
together in the inactive form.
• Certain Gα subunits interact with certain
enzymes. For example, Gs interacts with
adenylyl cyclase as described above.
• Gα subunits are distinguished from each other
by subscripts including s, i, and q (Gαs, Gαi,
and Gαq).
• The identity of the enzyme determines which
second messengers will be produced (or
inhibited). Adenylyl cyclase and phospholipase
C are two enzymes regulated by G proteins
that are responsible for regulating messengers
with important signaling roles.
Heterotrimeric G proteins
A. Adenylyl cyclase
• Two different Gα proteins regulate the activity of
adenylyl cyclase; the Gαs system stimulates its
activity while the Gαi inhibits it.
• Epinephrine (adrenaline) is a hormone that signals
with cAMP as the second messenger. In liver,
muscle, and adipose cells, the biological response
that results is the breakdown of stored
carbohydrates (glycogen) and fat for use as energy.
(Glucagon is a hormone that also stimulates
glycogen breakdown in liver).
• In the heart, the number of beats per minute (heart
rate) is increased by this signaling process.
1. Gαs
• The active Gα stimulates adenylyl cyclase
produce the second messenger cAMP.
• The enzyme phosphodiesterase converts cAMP
to 5′-AMP, ensuring that the amount of cAMP
in the cell is low.
• cAMP activates cAMP-dependent protein
kinase A, known as protein kinase A (PKA).
The activation process involves cAMP binding
to the regulatory or R subunits of PKA,
enabling the release of catalytic or C subunits.
Freed C subunits of PKA are active.
• PKA phosphorylates its protein substrates,
many of which are enzymes, on serine and
threonine residues.
• Phosphorylation regulates the activity of
proteins and enzymes and can lead to
intracellular effects.
• Protein phosphatases can dephosphorylate the
phosphorylated proteins to regulate their
activity.
• Over time, the Gαs will hydrolyze GTP to GDP
to terminate the activation of adenylyl cyclase
and the production of cAMP.
Activation of PKA by cAMP.
2. Gαi
• When Gαi is activated, it interacts with the
active adenylyl cyclase to inhibit its ability
to produce cAMP.
• In response, PKA will not be activated and
its substrates will not be phosphorylated.
B. Phospholipase C
• A variety of neurotransmitters, hormones,
and growth factors initiate signaling through
Gαq (Figure 17.7).
• After a hormone binds to its Gq-linked
receptor, the intracellular domain of the
occupied receptor interacts with Gq. The α
subunit of Gq releases GDP and binds GTP.
• The α subunit dissociates from the β and γ
subunits and then the α subunit activates
phospholipase C to cleave the membrane lipid
phosphatidylinositol 4,5-bisphosphate (PIP2).
Generation of second messengers in response to Gαq
activation of phospholipase C.
• The products of this cleavage are inositol 1,4,5trisphosphate (IP3), which is released into the
cytosol, and diacylglycerol (DAG), which
remains within the plasma membrane.
• IP3 binds to a specific receptor on the
endoplasmic reticulum, causing release of
sequestered calcium.
• Calcium and DAG together activate the
calcium-dependent protein kinase named
protein kinase C (PKC).
• IP3, DAG, and calcium are second messengers
in this system.
• PKC catalyzes phosphorylation of cellular proteins
that mediate cellular responses. Effects of
intracellular calcium are mediated by the calciumbinding protein calmodulin (Figure 17.8).
• After calcium is released from the endoplasmic
reticulum in response to the signaling of
hormones or neurotransmitters, the transient
increase in intracellular calcium concentration
favors formation of the calmodulin-calcium
complex.
• The calmodulin-calcium complex is an essential
component of many calcium-dependent enzymes.
Binding of the complex to inactive enzymes
results in their conversion to active enzymes.
Calmodulin mediates many effects
of intracellular calcium.
IV. RAS G PROTEINS
• Ras G proteins are homologous to the α subunits of
heterotrimeric G proteins. They do not regulate
membrane-bound enzymes or induce the
production of second messengers.
• Instead, their activation by GTP allows them to
initiate a cytoplasmic phosphorylation cascade that
termi-nates with activation of gene transcription.
• In this signaling scheme, Ras proteins are viewed as
relay switches between cell surface receptors and a
cascade of serine/threonine kinases that regulate
nuclear transcription factors. Such signaling is
important in the regulation of cell proliferation.
• The aberrant function of Ras proteins may
contribute to the malignant growth properties
of cancer cells.
A. Signaling mechanism
• Ras proteins are involved in signaling by
certain hormones and growth factors that are
ligands of catalytic receptors.
• A linear pathway from the cell surface to the
nucleus has been described, with Ras acting as
an intermediary.
• Ligand binding to catalytic receptors can cause
phosphorylation of tyrosine residues within
the receptors.
• The receptor’s phosphotyrosines provide
“docking” or binding sites for intracellular
adaptor proteins such as SHC and Grb2 that
contain regions known as SH2 domains.
• Ras-specific guanine exchange factor (GEF)
SOS joins the complex, followed by Ras. The
SHC-SOS-Ras complex exchanges GTP for
GDP on Ras, activating Ras.
• Ras-GTP promotes binding and
phosphorylation of Raf, a serine protein
kinase (also known as MAPKKK for mitogenactivated protein kinase kinase kinase).
• A phosphorylation cascade then includes
mitogen-activated protein kinases kinases
(such as MEK) that phosphorylate and
activate mitogen-activated protein kinase
(MAPK, aka extracellular signal-regulated
kinases or ERK), enabling it to translocate
to nucleus where it phosphorylates a
transcription factor (such as ELK).
• The cascade terminates with transcription
of genes for immediate early genes involved
in cell division. Hydrolysis of GTP to GDP
by RAS terminates the signaling process.
Ras signaling via activation of a cytoplasmic serine/threonine cascade.
• This linear pathway is now recognized to be
only a part of a very complex signaling
circuit in which Ras proteins are involved.
• Ras signaling involves a complex array of
pathways, where crosstalk, feedback loops,
branch points, and multicomponent
signaling complexes are seen.
B. Ras mutations and cell proliferation
• Mutations in Ras genes result in Ras proteins
that cannot hydrolyze GTP to GDP to
inactivate the signaling process.
• The Ras protein then remains in the active
state without stimulation of the receptor and
continues to send signals to induce
progression through the cell cycle. The result is
excessive cell proliferation that can lead to
malignancy.
Chapter Summary
• G proteins are intracellular signaling proteins
named for the ability to bind to and hydrolyze
GTP.
• Two categories of G proteins are described:
heterotrimeric G proteins that regulate second
messenger production and Ras superfamily small
G proteins.
• Heterotrimeric G proteins are composed of α, β,
and γ subunits and are activated by ligand binding
to G protein–linked receptors.
• Active G protein–linked receptors interact
with membrane-bound enzymes and
regulate their function.
• Products of reactions catalyzed by G
protein–linked enzymes are second
messengers that amplify the signal sent to
the cell by the ligand.
• Second messengers often regulate the
activity of certain serine/threonine protein
kinases.
• Adenylyl cyclase and phospholipase C are
enzymes regulated by G proteins.
• Adenylyl cyclase is regulated by Gs proteins that
stimulate its activity and Gi proteins that inhibit
its activity.
• cAMP is the second messenger whose production
is regulated by adenylyl cyclase. cAMP activates
PKA.
• Phospholipase C is activated by Gq proteins that
stimulate its activity to cleave the membrane lipid
PIP2.
• IP3 and DAG are products of this cleavage and are
the second messengers.
• IP3 induces the release of calcium from the
endoplasmic reticulum.
• Calcium and DAG activate PKC.
• Calcium binds to calmodulin which regulates the
activity of other proteins.
• The GTP-binding protein Ras is an intermediary in
signaling via some catalytic receptors.
• Activated Ras can stimulate the MAP kinase cascade
of serine/threonine phosphorylations that can result
in stimulation of gene transcription.
• Ras signaling is involved in the stimulation of cell
proliferation.
• Mutations in Ras can cause unregulated cell division
and malignancy.