molecular biology and pharmacology G protein, receptor, transport of drug ankoring protein, adapter protein, cell signal

1. Molecular Pharmacology
of
Cell Signaling
Mohanad AlBayati
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: aumnmumu@covm.uobaghdad.edu.iq
aumnmumu@yahoo.com

2. Previous soundness of drug cell
signal
• What is cell signaling?
• Signal transduction
• Receptors
• Types
• Functions
• Steps for signaling

5. - Guanlyl cyclase activity

7. Receptors
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.
Or
• Stop the binding of the native agent without eliciting a
response - an antagonist.
There are four ‘superfamilies’ of receptors.

8. . 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
thyroid hormones.
Receptors

9. Today soundness of drug cell
signal
 What is G protein coupled receptor
 Regulation
 What is G protein
 Regulation
 Mode of action

12. 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

13. Neurotransmitter receptors
Ligand – gated channels:
• Nicotinic acetylcholine receptor
• NMDA-type glutamate receptor
• Glycine receptor
• GABAA receptor
• Serotonin receptor (5-HT3)
G protein-coupled receptors:
• Muscarinic acetylcholine receptor (several types)
• Catecholamine receptors
• Histamine receptors (H1, H2)
• 5-HT receptors other than 5-HT3
• GABAB receptors
• ‘Metabotropic’ glutamate receptors
• Peptide receptors (Endorphin, cholecystokinin..)

23. `

43. Into nucleus

46. Thanks

47. The G Protein-Coupled Receptor
(GPCR) Superfamily
• 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
genes.
• Mammalian GPCRs: nearly 300 different kinds –
grouped into 3 main subfamilies:

48. • 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.

49. • GPCRs Interact guanine nucleotide-binding
proteins (aka G-proteins)
• Largest family of membrane proteins in the
human genome
• Eukaryotic trans membrane receptors
• Seven helices spanning the membrane

50.  Roles:
 - Light and smell processing
- Behavior and mood
- Immune response
- Autonomic nervous system
transmission
- Blood pressure
- Heart rate
- Digestive processes
- CRITICAL FACTOR IN MANY DISEASES!

51.  Five different classes (based on sequence and
function):
 - Class A: Rhodopsin-like receptors
- Class B: Secretin receptor family
- Class C: Metabotropic
glutamate/pheromone
- Class D: Fungal pheromone receptors
- Class E: Cyclyic AMP receptors

52. 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
homology:
- 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).

54. Regulation of G protein-coupled
receptor function
Desensitization/resensitization– a decrease in responsiveness
during continuous drug application or a right-shift in a drug
dose-response curve.
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
expression.
Long-term changes in [GPCR]s and [accessory proteins]s known
to be induced by chronic drug treatment and involved in
several pathologies.

55. Phosphorylation
2nd messenger kinase
G protein receptor kinase (GRK)
Arrestin
β-arrestin binding to phosphorylated GPCR is
required to decrease GTPase activity prior to
desensitization.
Receptor trafficking, internalization, and
recycling (covered earlier; see Protein
trafficking and LGIC slides).

56. 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
number.
e.g., antidepressants (e.g., fluoxetine) incr [5HT]synapse decr 5HT
receptor density.
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
sensitive factor).
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.

57. D D D D
α
α
β
α γ
(1) Agonist binding
and G protein
activation
(2) Phosphorylation
P P
(3) Arrestin
binding
Arrestin
P P
Arrestin
P P
Clathrin(4) Clustering in
clathrin-coated
pits
(5) EndocytosisEndosomes
Arrestin
P P
D
(7) Recycling
(6) Dissociation of agonist:
• Dephosphorylation
• Sorting between cycling
and lysosomal pathways
(8) Traffic to
lysosomes
Lysosomes
Mechanisms of Receptor Regulation

58. Another Receptor – G Protein Cycle

59. Structure, function and mechanisms
of G-Proteins

60. 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
Fig. 15.1
Examples of GTPase proteins
Ras, Cdc-42
(hormone, GF, drug)

61. 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.“

62. G-Protein = Guanine-nucleotide
binding protein
(GNBD)
1
2
5
4
3
Guanine
Ribose
Phosphates
α
1
3
42
6
5 7
89
Guanosine
EsterAnhydride
Guanosine-triphosphate - GTP

63. G-Protein families
• Heterotrimeric G-Proteins (Transducin, Gi, Gq …), 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
membranes
+ 60 further distinct families
Leipe et al., JMB (2002)

64. GTPases and disease.
• Damage to these small GTPase switches can have
catastrophic consequences for the cell and the
organism.
• 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
human cancers.

65. 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).

66. The heterotrimeric G proteins transmit signals
from a variety of cell surface receptors to enzymes
and channels
• Stimulated by receptors
• Act on effectors
• Regulated by nucleotide
exchange and hydrolysis

67. The signal is usually passed from a 7-helix receptor to an
intracellular G-protein.
 Seven-helix receptors are thus called GPCR, or G-
Protein-Coupled Receptors.
 Approx. 800 different GPCRs are encoded in the human
genome.

68.  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
epinephrine.

69. These domains include residues adjacent to the terminal
phosphate of GTP and/or the Mg++ associated with the
two terminal phosphates.
Inhibitory G
GTPS
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.

70. 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.
O
OHOH
HH
H
CH2
H
OPOPOP
O
O
O
O
O O
O
NH2
NH
NN
N
O
H O
H
GTP hydrolysis

71. 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
PDB 1GP2
G – face view of -propeller
PDB 1GP2

72. 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
neurons.
There is a larger family of small GTP-binding switch
proteins, related to G.

73. 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.

74. 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.
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase Activating
Proteins, promote GTP
hydrolysis.
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)

75.  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.
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)

76.  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.
protein-GTP (active)
GDP
GEF GAP
GTP Pi
protein-GDP (inactive)
GEFs, Guanine Nucleotide
Exchange Factors, promote
GDP/GTP exchange.

77.  &  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.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
 cytosol
GDP GTP
The  subunit of
a G-protein (G)
binds GTP, & can
hydrolyze it to
GDP + Pi.

79. The sequence of events by which a hormone activates
cAMP signaling:
1. Initially G has bound GDP, and ,, &  subunits
are complexed together.
G,, the complex of  &  subunits, inhibits G.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
 cytosol
GDP GTP

80. 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).
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
 cytosol
GDP GTP

81. 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.
AC
hormone
signal
outside
GPCR plasma
membrane
GTP GDP ATP cAMP + PPi
 cytosol
GDP GTP

82. Fig 15.3 The G Protein Cycle

83. GProtein-LinkedReceptors

84. GProtein-LinkedReceptors

85. GProtein-LinkedReceptors

86. GProtein-LinkedReceptors
note how activation is
reversible

87. GProtein-LinkedReceptors
the more ligand binding,
the more K+ in cytoplasm

88. 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
Gα subunit.
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.

89. 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
currents.

90. • 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)

91. • ACTH - Stimulates the synthesis and release of
Cortisol (zona fasiculata of adrenal cortex in
kidneys)
• 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

92. • 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

93. 
How G-protein-coupled receptors work (1)

extracellular space
cytosol
 heterotrimeric
G-protein
‘7TM’ - receptor
GDP
GDP
N
GTP
Ligand

94. How G-protein-coupled receptors work (2)
inactive

N
GDP

GTP
P

N
active

95. How G-protein-coupled receptors work (3)
ATP
inactive
inactive
active
cAMP
cAMP
Protein kinase A
Phosphorylation of multiple target proteins
 
GTP
active
Adenylate cyclase

96. Some G-proteins are inhibitory
-Adrenoceptor
2-Adrenoceptor
s
GTP
AC
active
AC
inactive
i
GTP

97. -Subunits of G proteins may have
regulatory activity, too
K+
Muscarinic (M2)
acetylcholine receptor
Kir

AC
inactive
i
GTP

98. G-proteins regulate diverse effector
systems
s
adenylate cyclase  protein kinase AcAMP 
i1
adenylate cyclase  protein kinase AcAMP 
q
phospholipase C 
PIP2 IP3 + DAG protein kinase C
phosphorylation of
multiple proteins
Ca++
ER
t cGMP phosphodiesterase  cGMP 

99. Many transmitters have multiple GPCR with different
downstream signaling mechanisms
Norepinephrine, 1 IP3 + DAG 
epinephrine2 cAMP 
1,2 cAMP 
Dopamine D2 - D4 cAMP 
D1, D5 cAMP 
Acetylcholine M1,,M4,M5IP3 + DAG 
M2, M3 cAMP 

100. Thanks