G protein-coupled receptors (GPCRs) are involved in many important physiological processes and are the targets of many drugs. They detect extracellular signals and activate intracellular effector proteins, usually via heterotrimeric G proteins. When a ligand binds to a GPCR, it causes a conformational change that allows the G protein's α subunit to exchange GDP for GTP. The GTP-bound α subunit then dissociates from the βγ complex and activates downstream effectors such as adenylyl cyclase. Eventually the Gα hydrolyzes GTP to GDP and reassociates with the βγ complex to terminate signaling. Rodbell and Gilman received the Nobel Prize for discovering GPCRs and their role in signal trans

1. Signal Sorting by G Protein
Linked Receptors
Major advisor:
Dr Jayakumar
Professor and Head
Dept of Vet Pharmacology & Toxicology
Speaker:
M.D Bayer Darmel
Sr MVSc Dept Pharmacology & Toxicology
receptor
tsqi
G protein
cAMPCa2+
intracellular
messenger
enzymechannel
effector

2. History
• Refers to a “receptive substance” describing the cellular
sites of interaction of drugs curare/nicotine and
atropine/pilocarpine in neuromuscularjunctions. (Langley. 1909)
• 1969: proposition of an intermediate transducer to link
distinct receptors to common effector adenylyl effector,
cyclase, and identification of the heterotrimeric G‐
protein,Gs.(John C. Foreman,.2003)
• 1983: rhodopsin was the first GPCR to be cloned
• The classical G protein signaling pathway that was identified very
early on was the activation of the adenylyl cyclase‐cAMP pathway
by G s (Gilman, 1987).
• Rodbell and Gilman were jointly awarded the Nobel Prize in 1994.
(John C. Foreman,.2003)
• 2000: first crystal structure of a GPCR(John C. Foreman,.2003)

3. Noble Prize for G PCR
Rodbell and Gilman were jointly awarded the Nobel Prize in 1994
for their discovery of G protein couple receptors and the role of these
protein in signal transduction in cell
Martin Rodbell, 1925–1998 USA
Alfred Goodman Gilman USA

4. G protein couple receptors
• G protein-coupled receptors (GPCRs),
also known as seven transmembrane
domain receptors, 7TM receptors,
heptahelical receptors, and G protein-
linked receptors (GPLR) .(David L et al,.2005)
• Also called metabotropic receptors and
serpentine receptors.(Miligan.1995 )

5. Importance
 G protein-coupled receptors are involved in many diseases,
and are also the target of around half of all modern medicinal
drugs.(Hardman et al,.2001)(Marchese et al.)
 G protein-coupled receptors are found only in eukaryotes,
including yeast(Saccharomyces cerevisiae)Saccharomyces cerevisiae), plants,
choanoflagellates, and animals
 Pathways involving these receptors are the targets of
hundreds of drugs, including antihistamines,
neuroleptics, antidepressants, and
antihypertensives(.(Miligan.1995 Ad.Ph V32)
• All GPCRs signal via the use of G-proteins

6. GPCRs are receptors for:
• Light, odours and gustative molecules
• Biogenic amines; dopamine, histamine, serotonin
• Eicosanoids .
• opioids,
• amino acids such as GABA, and many other
peptide and protein ligands
• Peptide and protein hormones
Panacreatic hormones
Gastrointestinal
Thyroid (Hardman,.2001)

7. Classification(I)
• Muscarinic acetylcholine receptors (several types)
• Catecholamine receptors
• Serotonin receptors 5-HT1,2,4,6
• GABA receptor
• Metabotropic’ glutamate receptors (11 subtypes)
• Purine receptors (P2Y): Adenosine, AMP, ADP,
ATP
• Peptide hormone receptors(Michael ,.2005)

8. Classification(II)
• Class A (or 1) (Rhodopsin-like)
• Class B (or 2) (Secretin receptor family)
• Class C (or 3) (Metabotropic
glutamate/pheromone)
• Class D (or 4) (Fungal mating pheromone
receptors)
• Class E (or 5) (Cyclic AMP receptors)
• Class F (or 6) (John C. 2000

9. John C. Foreman,.2003

10. The number of sequences in each subfamily

11. G-Protein-Coupled Receptors
John C. Foreman,et al,.2003

12. Physiological roles
 The visual sense, sense of smell and pheromones (vomeronasal receptors),
 Behavioral and mood regulation: receptors in the mammalian brain
( serotonin, dopamine)
 Regulation of immune system activity and inflammation (chemokines)
 Autonomic nervous system transmission: both the sympathetic and
parasympathetic nervous systems (blood pressure, heart rate and digestive
processes)
 The G-Protein-Coupled Receptor GCR1 Regulates DNA
Synthesis
 GPCRs comprise the largest family of cell surface receptors. In mice, there are 1000
different receptors involved in smell alone
 GPCR are able to regulate the rate of second messenger production or degration
 GPLR regulate ion flux through a battery oion chenall by direct GP regulation or via second
messenger.
• The human genome encodes morethan 1,000 members of this
family of receptors, specialized for transducing messages as
diverse as light, smells, tastes, and hormones (.(David L et
alt,.2005)

13. Ligand
• It is a signal triggering molecule binding to a site on a target
protein, by intermolecular forces such as ionic bonds,
hydrogen bonds and Van der Waals forces.
• Ligand can be selective for receptor or non selective .
• Ligands include substrates, inhibitors, activators, and
neurotransmitters.
• The affinity of ligand belongs to intermolecular force
• Can work as agonist or antagonist.(Miligan,1995)

14. Physiological role of GPCR
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15. Cell-to-cell communication by extracellular
signaling usually involves six steps
• (1) synthesis of the signaling molecule by the signaling cell
• (2) release of the signaling molecule by the signaling cell
• (3) transport of the signal to the target cell
• (4) detection of the signal by a specific receptor protein –
receptor-ligand specificity
• (5) a change in cellular metabolism, function, or development
= cellular respons.
• (6) removal of the signal, which usually terminates the
cellular response – degredation of ligand

17. Structure of GPCR
• Order of segments are
known
– N-terminus..
– Helix
– Intracellular loop
– Extracellular loop
– C-Terminus

18. Gether & Koblikas, 1998. JBC 273

19. GPCR cellular domains
• Extracellular domain
• By definition, a receptor's main function is to
recognize and respond to a specific ligand, for
example, a neurotransmitter or hormone
• Transmembrane domain
• Intracellular domain
• Adenylate Cyclase (AC) is a
transmembrane protein, with
cytosolic domains forming the
catalytic site.

20. Coupling to G protein
• Intracellular loop I3
– Main point of interaction
– 12 amino acids near N terminal of I3 mediates specificity (G protein subtype)
– Amino acids near C terminal of I3 mediate efficiency
– Varies in size between receptor subtypes
• Intracellular loop I2 (from TM3 to TM4)
• Mediates specificity and efficacy
• C terminal tail
– Determines efficiency
• Neurotransmitter interacting with amino acids in TM5 and TM6 transmit
conformation change to area of I3

21. G proteins(molecular switches)
• short for guanine nucleotide-binding proteins,
• G-proteins are heterotrimeric proteins composed of α (45 KDa), β (37
KDa), and γ (9 KDa) subunits (David L et alt,.2005)
• G-proteins interact with a receptor comprised of 7-membrane spanning
α-helices. Ligand binding induces. (Michael ,.2005)
Alpha
• Binds to guanosine nucleotides: GDP or GT
• four main families exist for Gα
subunits: Gαs
, Gαi
, Gαq/11
, and Gα12/13
.
(modified by attachment of fatty acid chain)
• Gαs
stimulates the production of cAMP from ATP.
• Gαi
inhibits the production of cAMP from ATP
• Gαq/11
stimulates membrane-bound phospholipase C beta, which
then cleaves PIP2
• Gα12/13
are involved in Rho family GTPase signaling

22. G Proteins
Beta and Gamma(CAAX)
• Five members of Beta subunit are identified(B1-B5)
• Binds to alpha subunit
• Stabilizes G protein in membrane
• Blocks alpha from interacting with effector
• Can be effectors
• The α and γ subunits have covalently attached lipid anchors, that
insert into the plasma membrane, binding a G-protein to the
cytosolic surface of the plasma membrane
• There is a larger family of small GTP-binding switch proteins,
• initiation & elongation factors (protein synthesis)
• Ras (growth factor signal cascades)
• Rab (membrane vesicle targeting and fusion)
• ARF (formation of vesicle coatomer coats)
• Ran (transport of proteins into & out of the nucleus)
• Rho (regulation of actin cytoskeleton

23. Lüllmann, Color Atlas of Pharmacology © 2000 Thieme

24. Types of G Protein Actions
• Indirect action
– G subunit activates enzyme
– Wide spatial extent due to diffusible second messenger
– Examples
• Adenylate cyclase
• Phospholipase C
• Phospholipase A2
• Phosphodiesterase
– Activates intracellular signalling pathways

25. Types of G Protein Actions
• Direct action
– G subunit directly gates channel
– Limited spatial extent
– Usually Gβγ
– Examples
• Stimulation of GIRK potassium channels
• Inhibition of calcium channels
• Regulation of Na-K pump
Lüllmann, Color Atlas of Pharmacology © 2000 Thieme

26. G-protein linked receptors
coupled to ion channels
• Acetylcholine (muscarinic)
• Adenosine & adenine nucleotides
• Adrenaline & noradrenaline
• Angiotensin
• Bombesin
• Bradykinin
• Calcitonin
• Cannabinoid
• Chemokine
• Cholecystokinin & gastrin
• Dopamine
• Endothelin
• Galinin
• GABA (GABAB)
• Glutamate (quisqualate)
• Histamine
• 5-Hydroxytryptamine (1,2)
• Leukotriene
• Melatonin
• Neuropeptide Y
• Neurotensin
• Odorant peptides
• Opioid peptides
• Platelet-activating factor
• Prostanoid
• Protease-activated
• Tachykinins
• Taste receptors
• VIP
• Vasopressin and oxytocin

27. G-protein activation
1. Initially the G-protein α subunit has bound GDP, and
the α, β, & γ subunits are complexed together. Gβ,γ
,
the complex of β & γ subunits, inhibits Gα
2. When the ligand binds to the GPCR it Altering the
conformation of the alpha subunit allows it to
exchange GDP for GTP.
3. Substitution of GTP for GDP causes another
conformational change in Gα
.
Gα
-GTP dissociates from the inhibitory βγ subunit
complex, and can now bind to and activate
Adenylate Cyclase
4. Adenylate Cyclase, activated by the stimulatory Gα
-
GTP, catalyzes synthesis of cAMP
5. Protein Kinase A (cAMP-Dependent Protein
Kinase) catalyzes transfer of phosphate from
ATP to serine or threonine residues of various
cellular proteins, altering their activity.
6. The complex of Gβ,γ
that is released when Gα
binds GTP is itself an effector that binds to
and activates or inhibits several other
proteins. For example, Gβ,γ
inhibits one of
several isoforms of Adenylate Cyclase,
contributing to rapid signal turnoff in cells that
Gprotn.gif

28. G protein Inactivation
1. Gα
hydrolyzes GTP to GDP + Pi
(GTPase). The
presence of GDP on Gα
causes it to rebind to the
inhibitory βγ complex. AdenylateCyclase is no
longer activated.
2. Phosphodiesterases catalyze hydrolysis of
cAMP to AMP.
3. Receptor desensitization varies with the
hormone.
In some cases the activated receptor is
phosphorylated via a G-protein Receptor
Kinase.
The phosphorylated receptor then may bind
to a protein β-arrestin
**GTPase activating proteins (GAPs), when
bound to the alpha subunit, enhance the
GTPase activity tremendously. GAPs are
critical negative regulators of G proteins.

29. The role of G Protein

30. There are three basic types of secondary
messenger molecules:
• Hydrophobic molecules: like diacylglycerol, IP3
,
and phosphatidylinositols, which are membrane-
associated and diffuse from the plasma membrane
into the space where they can reach and regulate
membrane-associated effector proteins
• Hydrophilic molecules: like cAMP, cGMP, and
Ca2+
, that are located within the cytosol
• Gases: nitric oxide (NO) and carbon monoxide
(CO), which can diffuse both through cytosol and
across cellular membranes.

31. Lüllmann, Color Atlas of Pharmacology © 2000 Thieme
The Stimulation of G Protein-Linked Signal
Transduction Pathways by α- and β-Adrenergic Receptors

32. Production of cAMP
1. Adenylyl cyclase produces cAMP by removing
two phosphate groups from ATP.
2. The phosphates are removed as pyrophosphate
(P-P).
3. Along with the removal of pyrophosphate the
molecule is cyclized.
4. cAMP phosphodiesterase then hydrolyzes
cAMP to AMP.
Cholera toxin: inactivates the GTPase activity of
the Gs alpha subunit, thereby keeping it active. This
causes oversecretion of chloride ions and water into
the gut (severe diahrrhea).
Pertussis toxin: this toxin inactivates the alpha
subunit of Gi. This blocks its ability to
negatively regulate its targets (whooping
cough).

33. Most effects of cAMP are mediated by protein kinase A (PKA)
1. PKA is activated by cAMP and mediates the majority of cAMP effects.
2. There are many different substrates of PKA in different cells, which may explain why rises
in cAMP in different cell types result in very different responses.
3. The inactive form of PKA is a heterotetramer of two catalytic (kinase) subunits and two
regulatory subunits.
4. The binding of cAMP to the regulatory subunits causes a conformational change that
releases the catalytic subunits.
5. The release of the catalytic subunits activates them, allowing them to phosphorylate their
substrates on serine and threonine residues.
33
Alberts 11-31
© Garland
serine
kinase
phosphatase
Residue in
target protein
N
H
CHC
CH2
O
O
-O OP
O
O
N
H
CHC
CH2
O
O
OH

34. Rapid and Slow responses to PKA
activation
**Some of the effects of PKA activation are rapid.
Example: the stimulation of glycogen breakdown to
glucose in muscle cells. This occurs by the direct
phosphorylation of proteins involved in glycogen
metabolism. This provides glucose for energy
production in muscle cells within seconds.
**Some of the effects of PKA are slower. Example: the
activation of gene expression, such as the
somatostatin hormone.
• Activated PKA can translocate into the nucleus.
• There it phosphorylates the transcription factor CREB
(cAMP response element binding protein).
3. When CREB is phosphorylated it binds to the cAMP
response element (CRE).
4. CREB-binding protein (CBP) binds to phosphorylated
CREB and activates transcription of genes that contain
CRE sequences, such as the somatostatin gene.
5. Many cAMP-induced genes contain CRE sequences
and are regulated by CREB and CBP.

35. Production of inositol phospholipids
**Phosphatylinositol (PI) 4-phosphate and PI 4,5 bisphosphate are produced by the sequential
actions of PI kinase and PIP kinase, respectively.
**PIs exist in the inner leaflet of the plasma membrane.
**PI 4,5 bisphosphate is especially important because its breakdown produces two different
second messengers.
**PI 4,5 bisphosphate is the least abundant of the PIs, and accounts for only 1% of total
phospholipids.
(Joyce J. Diwan.. 2008)

36. Phospholipase C-β is critical for
GPCR signaling
1. The G protein q (Gq) alpha subunit activates
the enzyme phospholipase C-β (PLC-β) in a
manner similar to how Gs activates adenylyl
cyclase.
2. Activated PLC-β cleaves PI 4,5 bisphosphate
to produce diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3).
3. Importantly, both DAG and IP3 are second
messengers that activate distinct intracellular
signaling molecules.
4. IP3 is a small, water-soluble molecule that
readily diffuses through the cytosol.
5. DAG remains embedded in the plasma
membrane, but like other PM lipids, can
diffuse laterally through the membrane.

37. The targets of IP3 and DAG
IP3: When IP3 diffuses to the membrane of the ER it binds to IP3-gated calcium release
channels (IP3Rs) in the membrane, triggering their opening.
**IP3Rs release calcium from the ER into the cytosol, rapidly increasing the concentration of
calcium in the cytosol. Ca2+
is perhaps the most common second messenger in cells.
**Calcium levels are quickly reduced by channels that pump it out of the cell, and by the
inactivation of IP3 by dephosphorylation and other means.
DAG: DAG has two signaling functions. First, it can be further cleaved to arachidonic acid,
which can initiate a complex cascade of lipid messengers. Second, DAG can activate a
serine/threonine kinase called protein kinase C (PKC).
**PKC requires both Ca2+
and DAG, along with membrane phospholipids, to be activated.
**PKC has numerous protein substrates that are unique from PKA.

38. Calmodulin
1. Calmodulin is a Ca2+
binding protein that has 4 high affinity binding sites for Ca2+
.
2. Calmodulin is extremely abundant in cells and accounts for as much as 1% of total protein.
3. Binding of calcium causes a conformational change in calmodulin.
4. At least two or more Ca2+
must bind before calmodulin changes conformations, making it
behave like a switch to increasing concentrations of calcium.
5. Calmodulin has no enzymatic function, and instead binds to target proteins and alters their
confirmation (as well as its own).
6. One of the most important group of calmodulin targets is the Ca2+
/calmodulin-dependent
protein kinases (CaM-kinases).

39. CaM-kinase II
1. CaM-kinase II is composed of a large
complex of about 12 subunits of CaM-
kinase II. For simplicity, only one is
shown here.
2. Upon Ca2+
/calmodulin binding, CaMKII
changes conformation and is activated.
3. Upon activation, CaMKII
autophosphorylates itself on an
autoinhibitory domain. This
phosphorylation event sustains CaMKII
activity without Ca2+
/calmodulin being
present in two ways. First, it locks
Ca2+
/calmodulin binding to it such that it
will not dissociate without the prolonged
return to normal calcium levels. Second,
it converts the enzyme to a calcium
independent form.
4. After this occurs, CaMKII can only be
inactivated if all of the subunits are
dephosphorylated by phosphatases
(overriding the CaMKII kinase activity).

40. GPCR desensitization
**Cells desensitize, or adapt, when exposed to high levels of ligand for a long period of time.
There are 3 mechanisms of desensitization at the level of the GPCR:
1. Receptor inactivation: The GPCR becomes modified such that it can no longer interact
with its G protein.
2. Receptor sequestration: The GPCR can be internalized and transported to an interior
compartment of the cell such that it no longer is exposed to ligand.
3. Receptor downregulation: The receptor can be degraded by lysosomes after it is
internalized.
G-protein-linked receptor kinases (GRKs): phosphorylate GPCRs upon their activation on
multiple serines and threonines.
This phosphorylation leads to binding of arrestin to the active GPCR. Arrestin triggers
desensitization by 1). inhibiting the binding of the G protein to the GPCR and 2). by
acting as an adaptor protein for the internalization of the receptor. Whether the receptor
is sequestered or degraded depends upon may factors.

41. Conclusion
■ A large family of plasma membrane receptors
with seven transmembrane segments act
through heterotrimeric G proteins. On ligand
binding, these receptors catalyze the exchange
of GTP for GDP bound to an associated G
protein, forcing dissociation of the subunit of
the G protein. This subunit stimulates or
inhibits the activity of a nearby membrane-bound
enzyme, changing the level of its second
messenger product.

42. Contin..
■
The cAMP produced by adenylyl cyclase is an
intracellular second messenger that stimulates
cAMP-dependent protein kinase,which mediates

43. Cont..
■ The cascade of events in which a single molecule
of hormone activates a catalyst that in turn
activates another catalyst, and so on,results in
large signal amplification; this is characteristic of
most hormone activated systems.
• Some receptors stimulate adenylyl cyclase
through Gs; others inhibit it through Gi. Thus
cellular [cAMP] reflects the integrated input of
two (or more) signals

44. Cont..
■ Cyclic AMP is eventually eliminated by cAMP
phosphodiesterase, and Gs turns itself off by
hydrolysis of its bound GTP to GDP.
When the epinephrine signal persists,
-adrenergic
receptor–specific protein kinase and arrestin 2
temporarily desensitize the receptor and cause it
to move into intracellular vesicles.
In some cases, arrestin also acts as a scaffold
protein, bringing together protein components of
a
signaling pathway such as the MAPK cascade

45. Cont..
■ Some serpentine receptors are coupled to a
plasma membrane phospholipase C that cleaves
PIP2 to diacylglycerol and IP3.
By opening Ca2 channels in the endoplasmic
reticulum, IP3 raises cytosolic [Ca2].
Diacylglycerol and Ca2 act together to activate
protein kinase C, which phosphorylates and
changes the activity of specific cellular proteins.
Cellular [Ca2] also regulates a number of other
enzymes, often through calmodulin.
T H E E N DT H E E N D