Download Signaling via G-Protein-Linked Cell


Signaling via G-Protein-Linked
Surface Receptors:
Cell-

This is largest family of cell-surface receptors

>100 members have already been defined in mammals

Though these receptors bind different hormones and
may mediate different cellular responses, they form a
class of receptors that functions similarly and exhibits
the following properties:

They consist of a single polypeptide chain that
threads back and forth across the lipid bilayer
seven times
Fig 15.17 Alberts 3rd Ed
 These seven α-helices contains ~ 22-24 hydrophobic
amino acid residues
 Four extra cellular loops and four intracellular loops
 Cytosolic loops and C-terminal segment, which face the
cytosol, are important for interaction with a Gprotein
Fig 20.10 Lodish 3rd Ed
 The signal transducing G-Protein associated with the
receptor functions as an on-off molecular switch,
which is in the off state when it binds GDP. Binding
of ligand to the receptor causes the G protein to release
its bound GDP and to bind GTP, converting the Gprotein to the on state
Fig 15.14 Alberts 3rd Ed
Fig 15.15 Alberts 3rd Ed
 The activated G-protein, with a bound GTP, binds to
and activates/inhibits an effector enzyme, which
catalyzes formation of 2nd messenger
 Hydrolysis of GTP, bound to the G-protein, switches
the G-protein back to the inactive i.e. in off state
 To illustrate the operation of this important class of receptors,
we will take example of epinephrine/nor-epinephrine receptors
and will try to understand structure-function relationship and
their associated signal-transducing G-proteins
 Effector system in this case is adenyl cyclase which
synthesizes cAMP as 2nd messenger

Binding of Epinephrine (EP) to β-and αAdrenergic Receptors Induces TissueSpecific Responses Mediated by cAMP:

Epinephrine and nor-epinephrine were originally
recognized as products of the medulla/core of the
adrenal gland

They are also called adrenaline and nor-adrenaline

Embryologically nerve cells drive from the same tissue
as adrenal medulla cells

They are also synthesized by neurons of central and
peripheral nervous system

Both hormones are charged compounds that belong
to the catecholamines, active amines containing the
compound catechol:
 Location:
Fig 25.9 Brum
 Synthesis:
Fig 24.10 Zubay
 In times of stress, such as fright or heavy exercise, all
tissues have an increased need for glucose and fatty
acids
 These principle metabolic fuels can be supplied to the
blood in seconds by the rapid breakdown of glycogen
in liver i.e. glycogenolysis and of triacylglycerol in
adipose storage cells i.e. lipolysis
Epinephrine was first hormone to
be isolate, characterized and
synthesized
Tyrosine hydroxylase:
The amount and activity are regulated by cAMPdependent mechanism that are responsive to
neurotransmitter (acetylcholine)
CaM kinase II:
-Present in all animal cells but especially
enriched in nervous system and highly
concentrated in synapses (neurotransmitters)
-Ca 2+ influx through voltage-gated Ca 2+
channel in plasma membrane stimulates cell
to secrete neurotransmitters
- Both secretion and resynthesis of the
neurotransmitters are stimulated when cell is
activated

In mammals, the liberation of glucose and fatty acids
can be triggered by:
 Binding of epinephrine and nor-epinephrine to βadrenergic receptors on the surface of hepatic and
adipose cells
 Epinephrine bound to similar β-adrenergic receptors on
heart muscle cells increases the contraction rate, which
increases the blood supply to the tissues
 Epinephrine bound to β-adrenergic receptors on smooth
muscle cells of the intestine causes them to relax
 Another type of adrenergic/epinephrine receptor, the αadrenergic receptor, is found on smooth muscle cells
lining the blood vessels in the intestine tract, skin and
kidneys
 Binding of epinephrine to α-adrenergic receptors
causes the arteries to constrict, cutting off circulation
to the peripheral organs
 All these diverse effects of one hormone are directed
common end:
to
a
 Supplying energy for the rapid movement of major
locomotor muscles in response to bodily stress
 All of the very different tissue-specific responses induced by
binding of epinephrine to β-adrenergic receptors are mediated
by a rise in the intracellular level of cAMP resulting from
activation of adenylate cyclase
 cAMP as a second messenger modifies the rate of different
enzyme catalyzed reactions in specific tissues generating
various metabolic responses
 Binding of numerous other hormones to their receptors also
leads to a rise in intracellular cAMP and characteristic tissuespecific metabolic responses
Table 20.5 Lodish 3rd Ed
 Two types of experiments have been used to establish the
identity of the β-adrenergic receptor
Fig 20.11 Lodish 3rd Ed
 Evidence that β-adrenergic receptor mediates induction of
epinephrine-initiated cAMP synthesis was studies with
receptors purified by affinity chromatography
Fig 20.12 Lodish 3rd Ed
Fig 2.17 Lodish 3rd Ed
Fig 15.4 Lodish 3rd Ed
 Similar results were obtained by transfecting cloned cDNA
encoding the β-adrenergic receptor into receptor-negative
cells

Analogs Provide Information about Essential
Features of Hormone Structure and are Useful as
Drugs:
 Studies with chemically synthesized analogs of epinephrine
and other natural hormones have provided additional
evidence that saturable cell-surface receptors are
physiologically relevant
 Analogs are of two kinds:

Agonists that mimic the function of a hormone
by binding to its receptor and cause the normal
response
 Antagonists that bind to the receptor but do not
activate hormone-induced effects

Therefore antagonist acts as an inhibitor of the natural
hormones/agonists by competing for binding sites on
the receptor, thereby blocking the physiological
response of the hormones

Comparison of the molecular structure and activity of
various catecholamine agonists and antagonists have
been used to define:
 the parts of the hormone molecule necessary for
binding to β-adrenergic receptors as well as
 the parts necessary for the subsequent induction of a
cellular response
Table 20.6 Lodish 3rd Ed/Table 20.2 Lodish 4th Ed
 Such studies indicate that the side chain containing the
NH groups determine the affinity of the ligand for
receptor while
 Catechol ring is required for the ligand - induces
increase in cAMP level

Two types of β-adrenergic receptors have been
identified in humans:
 Cardiac muscle cells possess β1-receptors which
promote increased heart rate and contractility by
binding catecholamines with the rank order of affinities
isoproterenol > norepinephrine > epinephrine

β-blockers like practolol are used to slow heart
contractions in the treatment of cardiac arrhythmias and
anginas


These β1-selective antagonists usually have little effect
on β1-adrenergic receptors on other cell types

The smooth muscle cells lining the bronchial passages
possess β2-receptors which mediate relaxation by
binding catecholamines with the rank order of affinities
isoproterenol >> epinephrine > norepinephrine

Agonist selective for β2-receptors like terbutaline, are
used in the treatment of asthma because they specifically
mediate opening of bronchioles, the small airways in the
lungs
Studies with Mutant β-adrenergic Receptors
Identify
Residues
that
Interact
with
Catecholamines:

Mutant forms of the β-adrenergic receptor generated by
site-specific mutagenesis

Expressed in culture cells and

Checked their ability to bind the agonist isoproterenol

Based on such studies, the model has been proposed
Fig 15.11 Lodish 6th Ed

Turned on to ill effect
Nature (1993) 365:603-604 (LH receptor-7A Talk)
LH Receptor 7A from Talk

Trimeric Signal -Transducing Gs Protein Links β Adrenergic Receptors and Adenylate Cyclase:

As explained, β-adrenergic receptors on different types
of mammalian cells mediate distinct tissue specific
responses, but the initial response following binding of
epinephrine is always the same i.e.

An elevation in the intracellular level of
cAMP

Such a rapid response requires balancing a rapid
synthesis of the molecules with its rapid
breakdown or removal
Fig 15.20 Alberts 3rd Ed

Increase in cAMP occurs as a result of
activation of adenylate cyclase a membrane
bound enzyme
Fig 15.21 Alberts 3rd Ed
Fig 13.14 Lodish 5th Ed

Cycling of Gs between Active and Inactive Forms:

A current model of how Gs couples receptor
activation to adenyl cyclase activation?
Fig 15.13 Lodish 6th Ed

Important evidence supporting this model has
come from studies with a non-hydrolysable
analog of GTP called GMPPNP in which a PNH-P replaces the terminal phosphodiester
bond in GTP
 Although this analog can not hydrolyzed, it binds to
Gsα like GTP
 The addition of GMPPNP and an agonist to an
erythrocyte membrane preparation results in a much
larger and longer-lived activation of adenylate cyclase
than occurs with an agonist and GTP
 Once GDP bound to Gsα is displaced by GMPPNP, it
remains permanently bound to Gsα
 Because the Gsα.GMPPNP complex is as functional as
the normal Gsα.GTP complex in activating adenylate
cyclase, as a result the enzyme is in a permanently
active state

Gsα Belongs to GTPase Super
Intracellular Switch Proteins:
Family
of

Understanding that how Gsα subunits cycle between
the active and inactive forms has come from studies of
a related and extremely important intracellular signal
transduction protein called Ras

Like Gsα, Ras alternates between an active on state
with bound GTP and inactive off state with a bound
GDP

In the on state Ras binds to and activates specific
effector proteins that control the growth and
differentiation of cells

Both Gsα and Ras are members of a family of
intracellular switch proteins collectively called as the
GTPase super family

Other members of this family include the Rabs
which regulate fusion of vesicles within the cells
Fig 16.43 Lodish 3rd Ed
Fig 14.20 Lodish 3rd Ed

Ras (~ 170 amino acids) is smaller than Gsα
(~ 300 amino acids) and its three dimensional
structure is similar to that of the part of Gsα that
binds GTP
Fig 5.18 Alberts 3rd Ed
Fig 5.20 Alberts 3rd Ed
- Abundant molecule in bacterial cell, where it serves an elongation factor in protein
synthesis, loading each amino-acyl tRNA molecule to ribosome.
- tRNA
molecule forms a tight complex with GTP-bound form EF-Tu

Cycling of Ras protein between the inactive formith
bound GDP and the active form with bound GTP
Fig. 20.17 Lodish 3rd Ed

Based on the similarity in the three dimensional
structure of Ras and the Gsα subunit of trimeric G
proteins and discovery of the role of GAP in cycling of
the Ras protein, a model of Gsα has been proposed
Fig. 20.18 Lodish 3rd Ed
Fig. 13.8 Lodish 5th Ed

Adenylate Cyclase is Stimulated and Inhibited by
Different Receptor-Ligand Complexes:
Fig 15.21 Lodish 6th Ed/Fig 20.20 Lodish 3rd Ed

Some Bacterial Toxins Irreversibly Modify G Proteins:

Confirmation of the GTP cycle came from a study of
certain bacterial toxins

The function of cholera toxin, a peptide produced by
the bacteria Vibrio cholerae was elucidated first

The classic symptom of cholera is massive diarrhea,
caused by water flow from the blood through the
epithelial cells into small intestine; death is often due to
dehydration
The study showed that cholera toxin irreversibly
activates adenylate cyclase in the intestinal epithelial
cells, causing a high level of cAMP


Later studies showed that the toxin irreversibly
activates adenylate cyclase in a large number of
cell types

Like diphtheria toxin, cholera toxin consists of two
types of peptide chains

One chain is enzyme that penetrates the cell surface
membrane and enters the cytosol, where it catalyzes
the covalent addition of an ADP-ribosyl group from
intracellular NAD+ to α-subunit of the Gs protein

This irreversibly modified Gs subunit can activate
adenylate cyclase normally but can not hydrolyze
bound GTP to GDP

Thus GTP remains bound to Gsα and Gs is always in
the activation mode: adenylate cyclase is continuously
turned on

As a result the level of cAMP in the cytosol rises
100-fold or more
Fig 19.17 Lodish 2nd Ed

In the intestinal epithelial cells, this rise apparently
causes certain membrane proteins to permit a massive
flow of H2O from the blood into intestinal lumen

Other bacterial toxin link ADP-ribose to other Gproteins and have proved invaluable in unraveling the
functions of these transducing molecules

For example, the pertussis toxin, secreted by the
“whooping cough” bacterium: Bordetella pertussis,
adds ADP-ribose to the α subunit of Gi

In this case, Giα linked ADP-ribose can not inactivate
adenylate cyclase

Pertussis toxin also adds ADP-ribose to and inactivates
the α subunits of several other G proteins
Table 19.6 Lodish 2nd Ed

Analogous Regions in All Seven-Spanning
Receptors Determine G-Protein and Ligand
Specificity:

Although G protein linked receptors are thought to
span the membrane seven times and hence their three
dimensional structures are predicted to be similar,
their amino acid sequences generally are quite
dissimilar
Fig 20.10 Lodish 3rd
Fig 15.11 Lodish 6th Ed

For example, the sequences of the closely related β1and β2-adrenergic receptors are only 50% identical

The sequence of the α- and β- adrenergic receptors
exhibit even less homology

The specific amino acid sequence of each receptor
determines which ligand it binds and which Gproteins interacts with

Studies with recombinant chimeric receptor proteins,
containing part of an α2 receptor and part of a β2receptor localized certain functional domains with
specific regions of receptor sequence
Fig 15.12 Lodish 6th Ed

Degradation of cAMP is also Regulated:



The level of cAMP usually controlled by the hormoneinduced activation of adenylate cyclase
Another point of regulation is the hydrolysis of cAMP
to 5’AMP by cAMP phosphodiesterase
This hydrolysis terminates the effect of hormone
stimulation
Fig 15.20 Alberts 3rd Ed

Many cAMP phosphodiesterases are activated by
increase in cytosolic Ca2+ which are often induced by
neuron or hormone stimulation

Role of cAMP in the Regulation of Cellular
Metabolism:

In earlier part, we saw that hormone stimulation of Gs
protein-linked cell-surface receptors leads to an
elevation of the level of cAMP

Recall that cAMP is the second messenger for many
hormones and that the effects of elevated cAMP differ
markedly in various types of cells
Table 20.5 Lodish 3rd Ed

Now, we will discuss how cAMP affects enzymatic
activity, thereby regulating cellular metabolism?

cAMP and Other Second Messengers Activate
Specific Protein Kinases:
 The diverse effect of cAMP are thought to be mediated
through the action of cAMP-dependent protein kinases
(cAPKs: also referred to as kinase A or PKA)
 These kinases catalyze transfer of the terminal phosphate
group from ATP to specific serine or threonine of selected
proteins
Fig 5.14 Alberts 3rd Ed
Fig 15.24 Alberts 3rd Ed
Fig 5.12 Alberts 3rd Ed
Fig 15.23 Lodish 6th Ed
 A-kinases are found in all animal cells and thought to
account for all the effects of cAMP in most of these cells
 The substrates for A-kinase differ in different cells types,
explaining why the effects of cAMP vary depending on the
target cell

Epinephrine Stimulates Glycogenolysis in Liver
and Muscle Cells:

As mentioned previously that cAMP-dependent protein
kinases induce many effects depending on the
particular substrate proteins that they phosphorylate

The first cAMP-mediated cellular response to be
discovered i.e. release of glucose from glycogen
(glycogenolysis) has been studied the most

This reaction occurs in muscle and liver cells
stimulated by epinephrine/agonist of β-adrenergic
receptors

Before describing how cAPKs regulate glycogen
metabolism? We first review the pathways of glycogen
synthesis and degradation
Fig 20.24 Lodish 3rd Ed







The fate of glucose 1-phosphate resulting from
degradation of glycogen differs in liver and muscle
cells
In muscle cells, glucose 1-phosphate produced from
glycogen is converted by phophogluco-mutase to
glucose 6-phosphate
This is metabolized via the Embden-Meyerhoff
glycolytic pathway to generate ATP which is source
of energy for contraction
In contrast, glucose from glycogen is not major
source of ATP in the liver
Rather, the liver stores and releases glucose
primarily for use by other tissues
Unlike muscle cells, liver cells contain a glucose 6phosphatase, which hydrolyzes glucose 6-phophate
to glucose
The free glucose is immediately released into the
blood and transported to other tissues, particularly
the muscles and brain

cAMP-Dependent Protein Kinase
the Enzymes of Glycogen Metabolism:

Regulates
In liver and muscle cells, the epinephrine-stimulated
elevation in the cAMP level enhances the conversation
of glycogen to glucose 1-phophate in two ways:

by inhibiting glycogen synthesis

by stimulating glycogen degradation
Fig 20.25 Lodish 3rd Ed
Fig 20.26 Lodish 3rd Ed
Fig 15.25 Alberts 3rd Ed
Fig 15.26 Alberts 3rd Ed

Kinase Cascade Permits Multi - Enzyme
Regulation and Amplifies Hormone Signal:

The
set
of
protein
phosphorylations
and
dephophorylations just described constitute a cascade, a
series of reactions in which the protein catalyzing one
step is activated or inhibited by the product of a
previous step

Although such a cascade may seem over-complicated,
it has at least two advantages for the cell:

First, a cascade allows an entire group of
enzyme catalyzed reaction to be regulated by a
single type of molecule

As we have seen, the three enzymes in the
glycogenolysis cascade:
•
cAMP-dependent protein kinase
•
Glycogen phosphorylase kinase and
• Glycogen phosphorylase
are regulated directly or indirectly by cAMP

•
•
Second, a cascade provides a huge amplification
of an initially small signal. For example:
Blood levels of epinephrine as low as 10-10 M
can stimulate glycogenolysis and release of
glucose resulting in an increases of blood
glucose levels by as much as 50%
An epinephrine stimulus of this magnitude
generates an intra-cellular cAMP concentration
of 10-6 M, an amplification of 104 M
•
Because three more catalytic steps precede the
release of glucose, another 104 M can occur
Fig 20.27 Lodish 3rd Ed
Fig 15.42 Alberts 3rd Ed
•
In striated muscle, the concentrations of the
three successive enzymes in the glycolytic
cascade – PKA, glycogen phosphorylase kinase
and glycogen phosphorylase - are in a 1:10:240
ratio, which dramatically illustrates the
amplification of the effects of epinephrine and
cAMP

Growth Hormone Cascade:
 The synthesis of many hormone is regulated by a cascade
of hormones
Fig 24.20 Zubay

Some G-Protein-Linked Receptors Activate the
Inositol Phospholipid Signaling Pathway by
Activating Phospholipase C-β
 Another crucial enzyme
Fig 19.4 Lodish 2nd Ed
 A role of phospholipids in signal transduction was first
suggested in 1953
 When it was found that some extracellular signaling
molecules stimulate the incorporation of radioactive
phosphate into phosphotidylinositol (PI), a minor
phospholipid in cell membrane

Later it was shown that this incorporation results from
the breakdown and subsequent synthesis of inositol
phospholipids

The inositol phospholipids found to be most important
in signal transduction were two phosphorylated
derivatives of PI:


PI phosphate (PIP)

PI biphosphate (PIP2)
These are thought to be located mainly in the inner
half of the plasma membrane lipid bilayer
Fig 15.29 Alberts 3rd Ed
Although PIP2 is less plentiful in animal cell
membranes than PI, it is the hydrolysis of PIP that
matters most
Fig 15.30 Alberts 3rd Ed

The chain events leading to PIP2 breakdown begins
with the binding of a signaling molecule of a Gprotein-linked receptor in the plasma membrane
Fig 15.33 Alberts 3rd Ed

More than 25 different cell-receptors have been shown
to utilize this transduction pathway
Table 20.7 Lodish 3rd Ed

We will first discuss how a rise in cytosolic Ca2+ ions
induces various metabolic responses and

Then consider how many hormones acting through IP3
cause this rise in the cytosolic Ca2+ level

We also examine the role of DAG in regulating other
cellular functions as all these 2nd messengers interact in
complex circuits to regulate crucial aspects of the grow
growth and metabolism of cells
Ionophores - An interesting group of low molecular
weight compounds (up to several
thousand daltons) compounds synthesized
by bacteria facilitates the translocation of
inorganic ions across membranes of other
cells