Cell signalling 2
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Transcript of Cell signalling 2
Cell signallingCell signalling
By: Khuram Aziz
M.phill biochemiatry
Previous discussionPrevious discussionWhat is cell signaling?Signal transductionReceptorsTypesFunctionsSteps for signaling
TodayTodayWhat is g protein coupled
receptorRegulationWhat is g proteinRegulatioonMode of action
Signal Reception: G Protein-Signal Reception: G Protein-Coupled ReceptorsCoupled 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 receptorsNeurotransmitter 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..)
The G Protein-Coupled Receptor The G Protein-Coupled Receptor (GPCR) Superfamily(GPCR) SuperfamilyLargest known receptor family –
Constitutes > 1% of the human 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.
GPCRsInteract 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 Almost all Receptors Comprise a Number of SubtypesNumber of Subtypes
Dopamine receptors - 5 subtypes5-HT receptors – 13 subtypesmGlu receptors - 8 subtypesAcetylcholine receptors – 5 subtypesIdentified 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 Regulation of G protein-coupled receptor functionreceptor 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
Arrestin
P PArresti
n
P 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 Another Receptor – G Protein CycleCycle
Structure, function and Structure, function and mechanisms of G-Proteinsmechanisms of G-Proteins
What are 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: active
2) 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-G-Protein = Guanine-nucleotide binding proteinnucleotide binding protein(GNBD)(GNBD)
12
5
43
Guanine
Ribose
Phosphates
α
1
3
42
65 7
89
Guanosine
EsterAnhydride
Guanosine-triphosphate - GTP
G-Protein familiesG-Protein familiesHeterotrimeric 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.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 regulatedG-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 The heterotrimeric G proteins transmit signals from a variety of transmit signals from a variety of cell surfacecell surface receptors to enzymes receptors to enzymes and channels 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 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.
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 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.
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 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
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.
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)
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)
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.
& 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 subunit of a G-protein (G) binds GTP, & can hydrolyze it to GDP + Pi.
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
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
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 + PP i
cytosol
GDP GTP
Fig 15.3Fig 15.3 The G Protein Cycle 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
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
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
How G-protein-coupled How G-protein-coupled receptors work (1)receptors work (1)
extracellular space
cytosol
heterotrimeric G-protein
‘7TM’ - receptor
GDP
GDP
N
GTP
Ligand
How G-protein-coupled How G-protein-coupled receptors work (2)receptors work (2)
inactive
N
GDP
GTP
P
N
active
How G-protein-coupled How G-protein-coupled receptors work (3)receptors work (3)
ATP
inactive
inactive
active
cAMPcAMP
Protein kinase A
Phosphorylation of multiple target proteins
GTP
active
Adenylate cyclase
Some G-proteins are Some G-proteins are inhibitoryinhibitory -Adrenoceptor
2-Adrenoceptor
s
GTP
ACactive
ACinactive
i
GTP
-Subunits of G proteins -Subunits of G proteins may have regulatory may have regulatory activity, tooactivity, too
K+
Muscarinic (M2)acetylcholine receptor
Kir
ACinactive
i
GTP
GG-proteins regulate diverse -proteins regulate diverse effector systemseffector systemss
adenylate cyclase protein kinase AcAMP
i1adenylate cyclase protein kinase AcAMP
qphospholipase C
PIP2 IP3 + DAG protein kinase C
phosphorylation ofmultiple proteins
Ca++
ER
tcGMP phosphodiesterase cGMP
Many transmitters have Many transmitters have multiple GPCR with different multiple GPCR with different downstream signaling downstream signaling mechanismsmechanisms
Norepinephrine, 1 IP3 + DAG epinephrine2 cAMP 1,2 cAMP
Dopamine D2 - D4 cAMP D1, D5 cAMP
Acetylcholine IP3 + DAG 2, M3 cAMP
Thanks