G-Protein Coupled ReceptorsAdenylyl Cyclase
Professor Dr Sagheer Ahmed
Shifa College of Pharmaceutical Sciences
Shifa Tameer-e-Millat University, Islamabad
RECEPTORS LINKED TO G PROTEINS
• G-protein–linked receptors constitute the largest family of receptors on
the cell surface, with more than 1000 members.
• These receptors mediate cellular responses to a diverse array of
signalling molecules, such as hormones, neurotransmitters, vasoactive
peptides, odorants, tastants, and other local mediators.
• Despite the chemical diversity of their ligands, most receptors of this
class have a similar structure.
• They consist of a single polypeptide chain with seven membrane-
spanning helical segments, an extracellular N terminus that is
glycosylated, a large cytoplasmic loop that is composed mainly of
hydrophilic amino acids between helices 5 and 6, and a hydrophilic
domain at the cytoplasmic C terminus.
GPCRs
• Most small ligands (e.g., epinephrine) bind in the plane of the
membrane at a site that involves several membrane-spanning
segments.
• In the case of larger protein ligands, a portion of the
extracellular N terminus also participates in ligand binding.
• The 5,6-cytoplasmic loop appears to be the major site of
interaction with the G protein, although the 3,4-cytoplasmic
loop and the cytoplasmic C terminus also contribute to
binding in some cases.
• G proteins couple cell-surface receptors to downstream
effectors.
G-Proteins, Small G-Proteins, Subunits
• G proteins are members of a superfamily of GTP-binding proteins.
• This superfamily includes the classic heterotrimeric proteins, as well as the so-called
small GTP-binding proteins, such as Ras.
• Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between
an active GTP-bound state and an inactive guanosine diphosphate (GDP)-bound
state.
• Heterotrimeric G proteins are composed of three subunits.
• At least 16 different α subunits (42 to 50 kDa), 5 β subunits (33 to 35 kDa), and 11 γ
subunits (8 to 10 kDa) are present in mammalian tissue.
• The αs-ubunits binds and hydrolyzes GTP, and also interacts with “downstream”
effector proteins such as adenylyl cyclase.
GTP-Hydrolyzing & Anchoring
• Historically, the α subunits were thought to provide the principal specificity to
each type of G protein, with the βγ complex functioning to anchor the trimeric
complex to the membrane.
• However, it is now clear that the βγ complex also functions in signal transduction
by interacting with certain effector molecules.
• Moreover, both the α and βγ subunits are involved in anchoring the complex to
the membrane.
• The α-subunit is held to the membrane by either a myristyl or a palmitoyl group,
whereas the βγ-subunit is held via a prenyl group.
Gs & Gi
• Because of the potential for several hundred combinations of the known subunits, G
proteins are ideally suited to link a diversity of receptors to a diversity of effectors.
• The many classes of G proteins, in conjunction with the presence of several receptor
types for a single ligand, provide a mechanism whereby a common signal can elicit the
appropriate physiological changes in different tissues.
• For example, when epinephrine binds adrenergic receptors in the heart, it stimulates
adenylyl cyclase, which increases heart rate and the force of contraction.
• However, in the periphery, epinephrine acts on adrenergic receptors that are coupled to
a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular
resistance and consequently increasing venous return and blood pressure.
Gs, Gi & Nobel Prize
• Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase.
• The heterotrimeric G protein known as Gs was so named because it stimulates adenylyl cyclase.
• A separate class of G proteins was given the name Gi because it is responsible for the hormonedependent inhibition of adenylyl cyclase.
• Identification of these classes of G proteins was greatly facilitated by the observation that the
subunits of individual G proteins are substrates for adenosine diphosphate (ADP) ribosylation
catalyzed by bacterial toxins.
• The toxin from Vibrio cholerae activates Gs, whereas the toxin from Bordetella pertussis
inactivates the cyclase-inhibiting Gi.
• For their work in identifying G proteins and elucidating the physiologic role of these proteins,
Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine
G-Protein Activation Follows a Cycle
• In their inactive state, heterotrimeric G proteins are a
complex of α βγ subunits in which GDP occupies the
guanine nucleotide binding site of the subunit.
• On ligand binding, the activated receptor interacts
with the α βγ heterotrimer to promote a
conformational change that facilitates the release of
bound GDP and simultaneous binding of GTP.
Cycle continues
• This GDP-GTP exchange stimulates dissociation of the
complex from the receptor and causes disassembly of
the trimer into a free subunit and βγ-complex.
• The free, active GTP-bound α-subunit can now interact
in the plane of the membrane with downstream
effectors such as adenylyl cyclase and phospholipases.
• Similarly, the βγ-subunit can now activate ion channels
or other effectors.
Cycle ends
• The α subunit terminates the signalling events that are
mediated by the α and βγ subunits by hydrolyzing GTP
to GDP and inorganic phosphate (Pi).
• The result is an inactive α -GDP complex that
dissociates from its downstream effector and
reassociates with a βγ subunit, thus completing the
cycle.
• The α subunit stabilizes -GDP and thereby substantially
slows the rate of GDP-GTP exchange and dampens
signal transmission in the resting state.
Activated Subunits Couple to a Variety of
downstream Effectors, Including Enzymes, Ion
Channels, and Membrane-TraffickingMachinery
• Activated α subunits can be coupled to a
variety of enzymes.
• On one hand, adenylyl cyclase, which is
activated by Gs, catalyzes the production of
cAMP from ATP.
• On the other hand, Gi inhibits adenylyl
cyclase and thus decreases [cAMP]i .
• Thus, different hormones—acting through
different G-protein complexes—can have
opposing effects on the same intracellular
messenger.
Phosphodiesterase
• G proteins can also activate enzymes that break
down cyclic nucleotides.
• For example, the G protein called transducin,
which plays a key role in phototransduction,
activates the cyclic guanosine monophosphate
(cGMP) phosphodiesterase, which catalyzes
the breakdown of cGMP to GMP.
• Thus, light leads to a decrease in [cGMP]i.
Phospholipase C
• G proteins can also be coupled to phospholipases., These enzymes catabolize phospholipids.
• The G-protein α q subunit activates phospholipase C
(PLC), which breaks phosphoinositol
bisphosphate (PIP2) into two intracellular
messengers, membrane-associated DAG
and cytosolic IP3.
• DAG stimulates protein kinase C (PKC),
• whereas IP3 binds to a receptor on the endoplasmic
• reticulum (ER) membrane and triggers the release of Ca from intracellular stores.
Ion Channels
• Some G proteins interact with ion channels.
• Agonists that bind to the -adrenergic receptor activate the L-type Ca
channel in the heart and skeletal muscle.
• The G protein Gs directly stimulates this channel as the alpha-subunit
of Gs binds to the channel, and Gs also indirectly stimulates this
channel via a signal-transduction cascade that involves cAMPdependent protein kinase.
The βγSubunits of G Proteins Can Also
Activate Downstream Effectors
• Considerable evidence now indicates that the βγ subunits can also interact with
downstream effectors.
• The neurotransmitter ACh released from the vagus nerve reduces the rate and
strength of heart contraction.
• This action in the atria of the heart is mediated by muscarinic M2 AChRs.
• These receptors can be activated by muscarine, an alkaloid found in certain
poisonous mushrooms.
• Muscarinic AChRs are very different from the nicotinic AChRs discussed earlier,
which are ligand-gated channels.
M2 Receptors & K Channels
• Binding of ACh to the muscarinic M2 receptor in the atria activates a heterotrimeric G
protein and liberates the βγ subunit complex.
• The βγ complex then interacts with a particular class of K channels, increasing their
permeability.
• This increase in K permeability keeps the Vm relatively negative, and thus renders the
cell more resistant to excitation.
• The βγ subunit complex also modulates the activity of adenylyl cyclase and PLC and
stimulates PLA2.
• Such effects of βγ can be independent of, synergize with, or antagonize the action of the
alpha subunit.
G-PROTEIN SECOND MESSENGERS:
CYCLIC NUCLEOTIDES
• Activation of Gs-coupled receptors results in the stimulation of adenylyl cyclase and a rise in intracellular
concentrations of cAMP.
• The downstream effects of this increase in [cAMP]i depend on the specialized functions that the responding
cell carries out in the organism.
• For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of
aldosterone and cortisol, whereas in the kidney, vasopressin-induced changes in cAMP levels facilitate
water reabsorption.
• Excess cAMP is also responsible for certain pathologic conditions.
• One is cholera.
• Another is McCune-Albright syndrome, which is characterized by short stature, subcutaneous ossification,
obesity, sexual precocity, and hyperfunction of multiple endocrine glands.
• This disorder is caused by a somatic mutation that constitutively activates the G protein s subunit.
cAMP & PKA
• cAMP exerts many of its effects through cAMPdependent protein kinase A (PKA).
• This enzyme catalyzes transfer of the terminal
phosphate of ATP to certain serine or threonine
residues within selected proteins. PKA is involved
in many cell-signalling pathways.
• To ensure firm regulation of phosphorylation
events, the cell tightly controls the activity of PKA
so that the enzyme can respond to subtle
variations in cAMP levels.
• One important control mechanism is the use of
regulatory subunits that constitutively inhibit PKA.
Regulation of PKA
• As mentioned previously, one important control mechanism is the use of
regulatory subunits that constitutively inhibit PKA.
• In the absence of cAMP, PKA is composed of four subunits—two regulatory and
two catalytic subunits so the complex has a low level of catalytic activity.
• Although most cells use the same catalytic subunit, different regulatory subunits
are found in different cell types.
• Binding of cAMP to the regulatory subunits induces a conformational change in
these proteins that diminishes their affinity for the catalytic subunits.
• Dissociation of the complex results in activation of the enzyme.
Further Regulation of PKA
• Another mechanism that contributes to regulation of PKA is the targeting
of the enzyme to specific subcellular locations.
• Such targeting promotes the preferential phosphorylation of substrates
that are confined to precise locations within the cell.
• PKA targeting is achieved by the association of a PKA regulatory subunit
with an A kinase anchoring protein (AKAP), which in turn binds to
cytoskeletal elements or to components of cellular subcompartments.
• Over 35 AKAPs are known.
PKA Regulation & AKAP
• The specificity of PKA targeting is highlighted by the observation that, in
neurons, PKA is localized to postsynaptic densities through its association
with AKAP79.
• This anchoring protein also targets calcineurin—a protein phosphatase—to
the same site.
• This targeting of both PKA and calcineurin to the same postsynaptic site
makes it possible for the cell to tightly regulate the phosphorylation state
of important neuronal substrates.
cAMP & Na Channel Activation
• The cAMP generated by adenylyl cyclase does not necessarily interact only
with PKA.
• For example, olfactory receptors interact with a member of the Gs family
called Golf.
• The rise in [cAMP] that results from activation of the olfactory receptor
activates a cation channel.
• Na influx through this channel leads to membrane depolarization and the
initiation of a nerve impulse.
cAMP, Protein Phosphorylation & Nobel Prizes
• For his work in elucidating the role played by cAMP as a second
messenger in signal transduction, Earl Sutherland received the 1971
Nobel Prize in Physiology or Medicine.
• In 1992, Edmond Fischer and Edwin Krebs shared the prize for their
part in demonstrating the role of protein phosphorylation in the
signal-transduction process.
Epinephrine Stimulates
Glycogen Breakdown
and Inhibits Glycogen
Synthesis via cAMP
Epinephrine & cAMP
• The importance of cAMP-mediated protein phosphorylation was first demonstrated for
glycogen catabolism in skeletal muscle.
• Glycogen, a glucose polymer stored primarily in liver and muscle cells, is the major
storage form of carbohydrate in the body.
• Epinephrine plays a major role in regulating both the synthesis and degradation of
glycogen.
• In muscle cells, for example, epinephrine induces glycogen breakdown and inhibits
glycogen synthesis by controlling a series of phosphorylation and dephosphorylation
events.
• The ultimate effect is release of glucose for use by the muscle cell.
PKA phosphorylates various enzymes
• Binding of epinephrine to its “adrenergic” receptor results in activation of PKA, which
phosphorylates three enzymes.
• First, PKA phosphorylates the regulatory subunits of inactive glycogen phosphorylase
kinase (PK), a massive enzyme.
• The net effect of this phosphorylation by PKA is to activate the enzyme, which allows PK
to phosphorylate a second inactive enzyme, glycogen phosphorylase b (GPb).
• The now-active glycogen phosphorylase a (GPa) then catalyzes the stepwise removal of
glucose 1-phosphate residues from glycogen.
• This intermediate is converted to glucose 6-phosphate, which in turn can enter the
glycolytic pathway.
PKA also activates a Phosphatase
• Second, PKA phosphorylates the active form of glycogen synthase (GS) and renders it inactive.
• Normally, the active GS transfers the glucose residue from uridine diphosphate (UDP)-glucose to a
free 4-OH group of a glucose residue at the end of the growing glycogen chain.
• Thus, phosphorylation of GS inhibits glycogen synthesis.
• Third, PKA also inactivates phosphoprotein phosphatase- 1 (PP1), the enzyme that is responsible
for removing the phosphates added in the PKA reactions just discussed.
• The mechanism of this inactivation is indirect: PKA phosphorylates and thus activates an inhibitor
of PP1, thus ensuring that the phosphate groups added to the other enzymes are not removed.
• The entire process is reversed when epinephrine is removed and the levels of cAMP fall.
Advantages of this phospho-, de-phosphorylation
• This coordinated set of phosphorylation and dephosphorylation reactions has several
physiological advantages.
• First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions.
• Second, it affords a large amplification to a small signal.
• The concentration of epinephrine needed to stimulate glycogenolysis in muscle is approximately
10-10 M.
• This subnanomolar level of hormone can raise [cAMP]i to approximately 10-6 M.
• As a consequence of the catalytic cascades, a further 10,000- fold amplification occurs, and
enough glucose is liberated to raise blood glucose levels from approximately 5 to 8 mM. Although
the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and
liver, cAMP-mediated activation cascades are used in the response of cells to a wide variety of
hormones.
Protein Phosphatases Reverse the Action of
Kinases
• One way that the cell can terminate a Camp signal is to use a phosphodiesterase to
degrade cAMP.
• In this way, the subsequent steps along the signalling pathway can also be terminated.
• However, because the downstream effects of cAMP usually involve phosphorylation at
serine and threonine residues of effector proteins, another powerful way to terminate
the action of cAMP is to dephosphorylate these effector proteins.
• Such dephosphorylation events are mediated by enzymes called serine/threonine
phosphoprotein phosphatases.
Types of Phosphatases
• Four groups of serine/threonine phosphoprotein phosphatases (PP) are known. 1, 2a, 2b, and 2c.
• These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine
residues.
• The balance between kinase and phosphatase activity plays a major role in the control of signalling events.
• PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to
epinephrine.
• PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the
action of other protein kinases.
• The Ca2-dependent PP2b—also known as calcineurin—is most prevalent in the brain.
• Calcineurin is also the target of the immunosuppressive reagents FK-506 and cyclosporine.
• PP2c appears to be of relatively minor importance.
• Growth factors often act via receptors that themselves are tyrosine kinases.
• That is, the receptor phosphorylates target proteins or themselves at tyrosine
residues rather than at serine or threonine residues.
• The enzymes that remove phosphates from these tyrosine residues are much
more variable than the serine and threonine phosphatases.
• The first phosphotyrosine phosphatase (PTP) to be characterized was the
cytosolic enzyme PTP1B from human placenta.
• PTP1B has a high degree of homology with CD45, a membrane protein that is
both a receptor and a phosphatase.
• cDNA sequence analysis has identified a large number of PTPs that can be
divided into two classes: membrane-spanning receptor-like proteins such as
CD45 and cytosolic forms such as PTP1B.
• A number of intracellular PTPs contain so-called Src homology-2 (SH2) domains,
a peptide se-quence or motif that interacts with phosphorylated tyrosine
groups.
• Several of the PTPs are themselves regulated by phosphorylation.
Thank You