Mohanad AbdulSattar Ali Al-Bayati, BVM&S, MSc. Physiol., PhD.
Assistant Professor of Pharmacology and Toxicology
Department of Physiology and Pharmacology
College of Veterinary Medicine
University of Baghdad
Al Ameria, Baghdad
Phone: 0964 7802120391
E. Mail: email@example.com
Previous soundness of drug cell
• What is cell signaling?
• Signal transduction
• Steps for signaling
Receptors are the sites at which biomolecules such as
hormones, neurotransmitters and the molecules responsible
for taste and odour are recognised.
A drug that binds to a receptor can either:
• Trigger the same events as the native ligand - an agonist.
• Stop the binding of the native agent without eliciting a
response - an antagonist.
There are four ‘superfamilies’ of receptors.
. These have 4 or 5 membrane-spanning helical subunits.
Their N- and C-terminii are found in the extracellular fluid. This family
includes ion channels.
. These have 7 helical transmembrane regions. Their N-
terminal is extracellular and the C-terminal in intracellular. This family
is coupled to the action of G-proteins: They are known as the G-
protein coupled receptors.
. These are tyrosine kinase-linked receptors with a single
transmembrane helix. The insulin and growth factor receptors fall
within this family.
. These receptors are found in the cell nucleus and are
transcription factors. They have looped regions held together by a
group of four cysteine residues coordinating to a zinc ion. These motifs
are called zinc fingers. The receptor ligands include steroids and
Today soundness of drug cell
What is G protein coupled receptor
What is G protein
Mode of action
G-protein-Coupled Receptors may dimerize or form
oligomeric complexes within the membrane.
Ligand binding may promote oligomerization, which may
in turn affect activity of the receptor.
Various GPCR-interacting proteins (GIPs) modulate
receptor function. Effects of GIPs may include:
altered ligand affinity
receptor dimerization or oligomerization
control of receptor localization, including transfer to
or removal from the plasma membrane
promoting close association with other signal proteins
The G Protein-Coupled Receptor
• Largest known receptor family –
Constitutes > 1% of the genome.
• Comprises receptors for a diverse array of molecules:
neurotransmitters, odorants, lipids, neuropeptides,
large glycoprotein hormones.
• Odorant receptor family alone contains hundreds of
• Mammalian GPCRs: nearly 300 different kinds –
grouped into 3 main subfamilies:
• Each GPCR family contains some orphan receptors,
which have been identified as members of the GPCR
superfamily by homology cloning but whose
activating ligand is unknown.
• But high throughput screening has recently added to
the advances in being able to identify the ligand.
• GPCRs Interact guanine nucleotide-binding
proteins (aka G-proteins)
• Largest family of membrane proteins in the
• Eukaryotic trans membrane receptors
• Seven helices spanning the membrane
- Light and smell processing
- Behavior and mood
- Immune response
- Autonomic nervous system
- Blood pressure
- Heart rate
- Digestive processes
- CRITICAL FACTOR IN MANY DISEASES!
Five different classes (based on sequence and
- Class A: Rhodopsin-like receptors
- Class B: Secretin receptor family
- Class C: Metabotropic
- Class D: Fungal pheromone receptors
- Class E: Cyclyic AMP receptors
Almost all Receptors Comprise a
Number of Subtypes
• Dopamine receptors - 5 subtypes
• 5-HT receptors – 13 subtypes
• mGlu receptors - 8 subtypes
• Acetylcholine receptors – 5 subtypes
• Identified by their pharmacological and functional
characteristics, rather than by strict sequence
- Some receptors for the same ligand show
remarkably little homology (e.g., histamine H3
and H4 have the lowest recorded homology (~ 20
%) to other histamine receptors H1 and H2).
Regulation of G protein-coupled
Desensitization/resensitization– a decrease in responsiveness
during continuous drug application or a right-shift in a drug
After removal of the drug, receptor activity recovers, although
the speed and extent of this resensitization can depend on
the duration of agonist activation.
Rapid desensitization (sec-min) results from receptor phos,
arrestin binding, and receptor internalization.
Long-term desensitization (down-regulation) involve changes in
receptor and/or G protein levels, and their mRNA stability and
Long-term changes in [GPCR]s and [accessory proteins]s known
to be induced by chronic drug treatment and involved in
2nd messenger kinase
G protein receptor kinase (GRK)
β-arrestin binding to phosphorylated GPCR is
required to decrease GTPase activity prior to
Receptor trafficking, internalization, and
recycling (covered earlier; see Protein
trafficking and LGIC slides).
Mechanisms of long-term down regulation
Long-term (> 1 hr) treatment with agonist induces the loss of total
cellular receptor number in addition to the decr in surface receptor
e.g., antidepressants (e.g., fluoxetine) incr [5HT]synapse decr 5HT
Receptor endocytosis: C-terminal domain determines whether they
enter the recycle pathway or the lysosomal pathway:
- 2 distinct motifs:
1. PDZ-domain interats with NHERF in a phos-dependent manner.
2. A short sequence that interacts with NSF (N-ethylmaleimide
Arrestin has also been shown to be important for recycling:
e.g., V2 vasopressin receptor, which continues to bind arrestin while in
endosomes, does not recycle back to plasma membrane.
D D D D
(1) Agonist binding
and G protein
Clathrin(4) Clustering in
(6) Dissociation of agonist:
• Sorting between cycling
and lysosomal pathways
(8) Traffic to
Mechanisms of Receptor Regulation
Structure, function and mechanisms
What are G-proteins?
• G proteins bind GTP: guanosine triphosphate. Control and amplify
intracellular signaling pathways
Exist in two states 1) bound GTP: active
2) bound GDP: inactive
Examples of GTPase proteins
(hormone, GF, drug)
1994 Nobel Prize in Medicine, Alfred Gilman and
Martin Rodbell, for their „discovery of G-Proteins
and the role of these proteins in signal
transduction in cells.“
• Heterotrimeric G-Proteins (Transducin, Gi, Gq …), in
7-TM receptor signalling
• Initiation, elongation, termination factors in protein
synthesis (IF1, EF-Tu, EF-TS)
• Signal recognition particle (SRP) and its receptor,
translocation of nascent polypeptide chains in the ER
• Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl,
Sar), molecular switches in signal transduction
• Dynamin superfamily of GTPases, remodelling of
+ 60 further distinct families
Leipe et al., JMB (2002)
GTPases and disease.
• Damage to these small GTPase switches can have
catastrophic consequences for the cell and the
• Several small GTPases of the Rac/Rho subfamily are
direct targets for clostridial cytotoxins.
• Further, Ras proteins are mutated to a constitutively-
active (GTP-bound) form in approximately 20% of
G-proteins are tightly regulated
3 types of accessory proteins that modulate cycling
of G-proteins between GTP/GDP
1. GAPs: GTPase-activating proteins. Stimulate GTP hydrolysis.
Inactivate G-protein. Example of a GAP: PLC.
2. GEFs: Guanine nucleotide-exchange factors: G-protein-coupled
receptors (GPCR). Stimulate dissociation of GDP (inactive) from
G-protein so GTP can bind (active).
3. GDIs: Guanine nucleotide-dissociation inhibitors. Inhibit release
of bound GDP (maintain G-protein in inactive state).
The heterotrimeric G proteins transmit signals
from a variety of cell surface receptors to enzymes
• Stimulated by receptors
• Act on effectors
• Regulated by nucleotide
exchange and hydrolysis
The signal is usually passed from a 7-helix receptor to an
Seven-helix receptors are thus called GPCR, or G-
Approx. 800 different GPCRs are encoded in the human
G-proteins are heterotrimeric, with 3 subunits , , .
A G-protein that activates cyclic-AMP formation within
a cell is called a stimulatory G-protein, designated Gs
with alpha subunit Gs.
Gs is activated, e.g., by receptors for the hormones
epinephrine and glucagon.
The -adrenergic receptor is the GPCR for
These domains include residues adjacent to the terminal
phosphate of GTP and/or the Mg++ associated with the
two terminal phosphates.
PDB 1GIAStructure of G proteins:
The nucleotide binding site
in G consists of loops that
extend out from the edge of
a 6-stranded -sheet.
Three switch domains have
been identified, that change
position when GTP
substitutes for GDP on G.
GTP hydrolysis occurs by nucleophilic attack of a water
molecule on the terminal phosphate of GTP.
Switch domain II of G includes a conserved glutamine
residue that helps to position the attacking water
molecule adjacent to GTP at the active site.
The subunit of the heterotrimeric G Protein has a
-propeller structure, formed from multiple repeats of a
sequence called the WD-repeat.
The -propeller provides a stable structural support for
residues that bind G.
It is a common structural motif for protein domains
involved in protein-protein interaction.
G - side view of -propeller
G – face view of -propeller
The family of heterotrimeric G proteins includes also:
transducin, involved in sensing of light in the retina.
G-proteins involved in odorant sensing in olfactory
There is a larger family of small GTP-binding switch
proteins, related to G.
Small GTP-binding proteins include (roles indicated):
initiation & elongation factors (protein synthesis).
Ras (growth factor signal cascades).
Rab (vesicle targeting and fusion).
ARF (forming vesicle coatomer coats).
Ran (transport of proteins into & out of the nucleus).
Rho (regulation of actin cytoskeleton)
All GTP-binding proteins differ in conformation
depending on whether GDP or GTP is present at their
nucleotide binding site.
Generally, GTP binding induces the active state.
A GAP may provide an essential active site residue, while
promoting the correct positioning of the glutamine
residue of the switch II domain.
Frequently a (+) charged arginine residue of a GAP
inserts into the active site and helps to stabilize the
transition state by interacting with () charged O atoms
of the terminal phosphate of GTP during hydrolysis.
proteins depend on
GAPs, GTPase Activating
Proteins, promote GTP
G of a heterotrimeric G protein has innate capability
for GTP hydrolysis.
It has the essential arginine residue normally provided
by a GAP for small GTP-binding proteins.
However, RGS proteins, which are negative regulators
of G protein signaling, stimulate GTP hydrolysis by G.
An activated receptor (GPCR) normally serves as GEF
for a heterotrimeric G-protein.
Alternatively, AGS (Activator of G-protein Signaling)
proteins may activate some heterotrimeric G-proteins,
independent of a receptor.
Some AGS proteins have GEF activity.
GEFs, Guanine Nucleotide
Exchange Factors, promote
& subunits have covalently attached lipid anchors that
bind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with
cytosolic domains forming the catalytic site.
GTP GDP ATP cAMP + PPi
The subunit of
a G-protein (G)
binds GTP, & can
hydrolyze it to
GDP + Pi.
The sequence of events by which a hormone activates
1. Initially G has bound GDP, and ,, & subunits
are complexed together.
G,, the complex of & subunits, inhibits G.
GTP GDP ATP cAMP + PPi
2. Hormone binding, usually to an extracellular domain
of a 7-helix receptor (GPCR), causes a conformational
change in the receptor that is transmitted to a G-protein
on the cytosolic side of the membrane.
The nucleotide-binding site on G becomes more accessible
to the cytosol, where [GTP] > [GDP].
G releases GDP & binds GTP (GDP-GTP exchange).
GTP GDP ATP cAMP + PPi
3. Substitution of GTP for GDP causes another
conformational change in G.
G-GTP dissociates from the inhibitory complex & can
now bind to and activate Adenylate Cyclase.
GTP GDP ATP cAMP + PPi
note how activation is
the more ligand binding,
the more K+ in cytoplasm
Regulation at the G protein level
Regulator of G protein signaling (RGS = GAPs = GTPase
activating proteins) family of proteins (> 20
members) regulate the rate of GTP hydrolysis in the
Can also attenuate G protein actions that are mediated
by βγ subunits, because they can alter the number of
βγ available by enhancing the affinity of Gα subunits
for the βγ after GTP hydrolysis incr rate of
reformation of the heterotimer.
Regulation at the G protein level (cont’d)
RGS proteins also important in regulating the temporal
characteristics of G protein actions.
E.g., RGS proteins accelerate the decay of agonist-
induced activation of GIRK (G protein regulated
inward rectifying K channels).
E.g., RGS proteins accelerate desensitization of
adrenergic receptor-induced N-type Ca2+ channel
• ADH - Promotes water retention by
the kidneys (V2 Cells of Posterior Pituitary)
• GHRH - Stimulates the synthesis and release of
GH (Somatotroph Cells of Anterior Pituitary)
• GHIH - Inhibits the synthesis and release of GH
(Somatotroph Cells of Anterior Pituitary)
• CRH - Stimulates the synthesis and release of
ACTH (Anterior Pituitary)
• ACTH - Stimulates the synthesis and release of
Cortisol (zona fasiculata of adrenal cortex in
• TSH - Stimulates the synthesis and release of a
majority of T4 (Thyroid Gland)
• LH - Stimulates follicular maturation and
ovulation in women; Stimulates testosterone
production and spermatogenesis in men
• FSH - Stimulates follicular development in women;
Stimulates spermatogenesis in men
• PTH - Increases blood calcium levels (PTH1 Receptor:
Kidneys and Bone; PTH2 Receptor: Central Nervous
system, Bones, Kidneys, Brain)
• Calcitonin - Decreases blood calcium levels (Calcitonin
Receptor: Intestines, Bones, Kidneys, Brain)
• Glucagon - Stimulates glycogen breakdown (liver)
• hCG - Promotes cellular differentiation; Potentially
involved in apoptosis
How G-protein-coupled receptors work (1)
‘7TM’ - receptor
How G-protein-coupled receptors work (2)
How G-protein-coupled receptors work (3)
Protein kinase A
Phosphorylation of multiple target proteins
Some G-proteins are inhibitory
-Subunits of G proteins may have
regulatory activity, too
G-proteins regulate diverse effector
adenylate cyclase protein kinase AcAMP
adenylate cyclase protein kinase AcAMP
PIP2 IP3 + DAG protein kinase C
t cGMP phosphodiesterase cGMP
Many transmitters have multiple GPCR with different
downstream signaling mechanisms
Norepinephrine, 1 IP3 + DAG
Dopamine D2 - D4 cAMP
D1, D5 cAMP
Acetylcholine M1,,M4,M5IP3 + DAG
M2, M3 cAMP