Molecular Pharmacology of

Cell Signaling

Mohanad AlBayati Mohanad AbdulSattar Ali Al-Bayati, BVM&S, MSc. Physiol., PhD.

Assistant Professor of Pharmacology and ToxicologyDepartment of Physiology and Pharmacology

College of Veterinary MedicineUniversity of Baghdad

Al Ameria, Baghdad Phone: 0964 7802120391

E. Mail: aumnmumu@covm.uobaghdad.edu.iqaumnmumu@yahoo.com

Previous soundness of drug cell signal

• What is cell signaling?• Signal transduction• Receptors• Types• Functions• Steps for signaling

- Guanlyl cyclase activity

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.

• Type 1. 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.

• Type 2. 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.

• Type 3. These are tyrosine kinase-linked receptors with a single transmembrane helix. The insulin and growth factor receptors fall within this family.

• Type 4. 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

Today soundness of drug cell signal

What is G protein coupled receptor

RegulationWhat is G proteinRegulationMode of action

Signal Reception: G Protein-Coupled Receptors

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

Neurotransmitter receptorsLigand – 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..)

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Into nucleus

Thanks

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:

• 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 human genome

• Eukaryotic trans membrane receptors• Seven helices spanning the membrane

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!

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

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).

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.

Phosphorylation2nd messenger kinaseG protein receptor kinase (GRK)Arrestinβ-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).

Mechanisms of long-term down regulationLong-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.

D D D D

αα

βα γ

(1) Agonist bindingand G protein

activation

(2) Phosphorylation

P P

(3) Arrestinbinding

ArrestinP P

ArrestinP P

Clathrin(4) Clustering inclathrin-coated

pits

(5) EndocytosisEndosomes

ArrestinP P

D

(7) Recycling

(6) Dissociation of agonist:• Dephosphorylation• Sorting between cycling

and lysosomal pathways

(8) Traffic tolysosomes

Lysosomes

Mechanisms of Receptor Regulation

Another Receptor – G Protein Cycle

Structure, function and mechanisms of G-Proteins

What are G-proteins?• G proteins bind GTP: guanosine triphosphate. Control and amplify

intracellular signaling pathways

Exist in two states 1) bound GTP: active2) bound GDP: inactive

Fig. 15.1

Examples of GTPase proteinsRas, Cdc-42

(hormone, GF, drug)

1994 Nobel Prize in Medicine, Alfred Gilman and Martin Rodbell, for their „discovery of G-Proteinsand the role of these proteins in signal transduction in cells.“

G-Protein = Guanine-nucleotide binding protein(GNBD)

12

5

43

Guanine

Ribose

Phosphates

α

1

3

42

65 7

89

Guanosine

EsterAnhydride

Guanosine-triphosphate - GTP

G-Protein families• 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

membranes + 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 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.

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 .b

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 and channels

• Stimulated by receptors• Act on effectors• Regulated by nucleotide

exchange and hydrolysis

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.

G-proteins are heterotrimeric, with 3 subunits a, b, g.

A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gsa.

Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon.

The b-adrenergic receptor is the GPCR for epinephrine.

These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg++ associated with the two terminal phosphates.

Inhibitory G

GTPS

PDB 1GIA Structure of G proteins:

The nucleotide binding site in Ga consists of loops that extend out from the edge of a 6-stranded b-sheet.

Three switch domains have been identified, that change position when GTP substitutes for GDP on Ga.

GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP.

Switch domain II of Ga 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

The b subunit of the heterotrimeric G Protein has a b-propeller structure, formed from multiple repeats of a sequence called the WD-repeat. The b-propeller provides a stable structural support for residues that bind Ga.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

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 Ga.

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.

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)

Ga 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 Ga.

protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)

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.

a & g 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 + PP i

cytosol

GDP GTP

The asubunit of a G-protein (Ga) binds GTP, & can hydrolyze it to GDP + Pi.

The sequence of events by which a hormone activates cAMP signaling:

1. Initially Ga has bound GDP, and a,b, &g subunits are complexed together.

Gb,g, the complex of b & g subunits, inhibits Ga.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

cytosol

GDP GTP

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 Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Ga releases GDP & binds GTP (GDP-GTP exchange).

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PP i

cytosol

GDP GTP

3. Substitution of GTP for GDP causes another conformational change in Ga.

Ga-GTP dissociates from the inhibitory bg complex & can now bind to and activate Adenylate Cyclase.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PP i

cytosol

GDP GTP

Fig 15.3 The G Protein Cycle

G P

rote

in-L

inke

d R

ecep

tors

G P

rote

in-L

inke

d R

ecep

tors

G P

rote

in-L

inke

d R

ecep

tors

G P

rote

in-L

inke

d R

ecep

tors

note how activation is reversible

G P

rote

in-L

inke

d R

ecep

tors

the more ligand binding, the more K+ in cytoplasm

Regulation at the G protein levelRegulator 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.

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.

• 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 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

• 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

bg

How G-protein-coupled receptors work (1)

a

extracellular space

cytosol

abg heterotrimeric G-protein

‘7TM’ - receptor

GDP

GDP

N

GTP

Ligand

How G-protein-coupled receptors work (2)

inactive

abg

N

GDP

a

GTP

P

bg

N

active

How G-protein-coupled receptors work (3)

ATP

inactive

inactive

activecAMP

cAMP

Protein kinase A

Phosphorylation of multiple target proteins

bg aGTP

active

Adenylate cyclase

Some G-proteins are inhibitoryb-Adrenoceptor

a2-Adrenoceptor

as

GTP

ACactive

ACinactive

ai

GTP

bg-Subunits of G proteins may have regulatory activity, too

K+

Muscarinic (M2)acetylcholine receptor

Kir

bg

ACinactive

ai

GTP

Ga-proteins regulate diverse effector systems

asadenylate cyclase protein kinase AcAMP

ai1adenylate cyclase protein kinase AcAMP

aqphospholipase C

PIP2 IP3 + DAG protein kinase C

phosphorylation ofmultiple proteins

Ca++

ER

atcGMP phosphodiesterase cGMP

Many transmitters have multiple GPCR with different downstream

signaling mechanismsNorepinephrine, a1 IP3 + DAG epinephrinea2 cAMP b1,b2 cAMP

Dopamine D2 - D4 cAMP D1, D5 cAMP

Acetylcholine M1,,M4,M5IP3 + DAG M2, M3 cAMP

Thanks