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Signal Reception: G ProteinCoupled Receptors
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..)
The G Protein-Coupled Receptor (GPCR)
Superfamily
• Largest 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:
Three Main Mammalian GPCR Subfamilies
• Rhodopsin-like group – includes most of the
GPCRs.
• Glucagon-like group.
• Metabotropic glutamate (mGlu) and GABAB
receptor family.
Three Main Mammalian GPCR Subfamilies
(cont’d)
• Grouped according to > 20 % sequence homology.
• Databases for the classification of receptors into
subfamilies, phylogenetic trees, chromosome
localization, ligand binding constants and receptor
mutations can be found at www.gpcr.org/7tm
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).
• 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.
Originally published in Science Express on 25 October 2007.
Paper version: Science 23 November 2007: Vol. 318. no. 5854, pp. 1258 - 1265.
High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor
Vadim Cherezov, Daniel M. Rosenbaum, Michael A. Hanson, Søren G. F. Rasmussen, Foon Sun Thian,
Tong Sun Kobilka, Hee-Jung Choi, Peter Kuhn, William I. Weis, Brian K. Kobilka,Raymond C. Stevens
Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors constitute the largest
family of eukaryotic signal transduction proteins that communicate across the membrane. We report the
crystal structure of a human β2-adrenergic receptor–T4 lysozyme fusion protein bound to the partial
inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a
human G protein–coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is
enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely
spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component
for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice.
Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in
rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in
using rhodopsin as a template model for this large receptor family.
Types of G Proteins and their 2nd Messenger
Pathways
α, β, γ Subunits
α Subunit (23 isoforms): contains the GTP/GDP binding site
is responsible for identity.
β (5 isoforms) and γ (12 isoforms): are identical or very
similar, interchangeable in vitro; most of them are
ubiquitously expressed; membrane anchored through
prenylation of Gβ.
Golf: expressed in olfactory bulb, coupled to PLCβ.
GT (transducin): is coupled to cGMP phosphodiesterase and is
expressed in the rod cells of the retina (these cells are
Inactivated by light!!): hν hits rhodopsin -> opsin is
activated -> facilitates GTP loading of GT -> activates
cGMP phosphodiesterase -> cGMP (keeps Na+ and Ca2+
channels open to cause depol -> nt release) -> converted to
5’GMP (inactive => channels closed => membrane
polarization => no nt released).
Receptor Family 1 – Rhodopsin Family
D
R
Y
TM7
Extracellular
TM6
C
TM5
C
TN4
N
TM3
TM2
TM1
Y Y
Lipid Bilaye
CC
Intracellular
C
Receptor-Ligand Interactions:
Rhodopsin-like Family
6
HO
7
O S
H
OHS
+N
D
1
3
2
4
5
Receptor Family 2 – Glucagon-like Family
• Structurally similar to that of Family 1, except that they
have a much larger N-terminal domain, which contains
multiple potential S-S bridges.
• Most of the ligands binding to these receptors are peptides
or glycoprotein hormones:
- 30-40 residues is typical.
- likely to interact with the receptor over
large surface areas.
Receptor Family 2 – Glucagon-like Family
N
TM7
TN4
D
R
Y
Extracellular
TM6
C
TM5
C
TM3
TM2
TM1
Y Y
Lipid Bilaye
CC
Intracellular
C
Receptor Family 3 – mGluR/GABAB Family
• Extremely large extracellular N-terminal ligand binding
domain.
• Highly conserved 3rd short intracellular loop.
• Shares only ~ 12 % sequence homology with that of Family 1,
but the overall transmembrane topology is similar.
• Forms dimers:
- GABAB receptor forms heterodimers between
GABABR1 and GABABR2 through coiled coil regions in the
C-terminal tails.
- This dimerization is required for efficient
cell surface expression and signalling.
- Metabotropic glutamate receptors dimerization is
stabelized by disulfide bonds in the N-terminal
extracellular domain.
Common Experimental Tools used to Study GPCRs
D
Drug binding
and G protein
activation
α
β α
GDP
α
γ
α
β α
α
γ
Reformation of
receptor G protein
complex
The G Protein Cycle
D
D
Dissociation of
receptor-G protein
complex
GTP
β
α
α
α
γ
GTP
Inactivation of
Gα through intrinsic
GTPase activity
Pi
α
α
GDP
Receptor-G protein Interactions
How are receptor-G protein interactions measured?
• Ligand-binding assays:
Low- affinity
High-affinity
R + G(GTP-δ-S)
RG(GDP)
GDP
GTPγS
Without GTP, both high- and low-affinity states are measured.
With GTP and Mg2+, only low-affinity state is measured, because
Agonist binding rapidly induces change from high- to low-affinity.
Receptor-G protein Interactions
Structural features of receptors involved in G protein
activation
How does agonist binding cause receptor
conformational change?
• Agonists vary in their binding affinity for the
GPCR = drug-receptor interaction.
• How well the drug causes a conformational
change in the receptor to activate G proteins =
efficacy.
• There are multiple agonists (partial, full) with
different binding affinities.
Receptor-G protein Interactions
Structural features of receptors involved in
G protein activation
• Mechanism of conformational change highly conserved.
• Constraining intermolecular interactions that keep receptors
preferentially silent in the absence of agonist: such as between
TM5 & TM6 and between TM3 & TM7.
• E.g., ‘DRY’ motif in TM3 (earlier).
• Upon receptor activation, the arg is protonated  adjacent
residues move  tilting the TM helix  incr exposes
previously hidden sequences, which interact with G protein.
• Much evidence for preceding.
• However, the exact aa sequence responsible for this has been
difficult to pinpoint.
Constitutive Activity
• Many receptors show constitutive activity even when
expressed at physiol levels (e.g., rat dopamine D1, rat, human
hist H2, human dopamine D3, and human 5-HT1A).
• Inverse agonists.
• Mutations have been identified that incr the basal activity w/o
affecting the ability of agonists to further activate the
receptors.
• These mutations affect stabilizing interactions between helices
that hold the receptor in an inactive state and those interfering
with these interactions
Multiple active conformations – stimulus trafficking
• There may be several active conformations, which are
induced by certain drugs = stimulus trafficking.
• Different drugs can promote distinct receptor
conformations, which interact with different G
proteins resulting in activation of distinct signaling
pathways.
• E.g., partial agonists at human 5HT2A and 5HT2C
receptors  differential stimulation of IP and AA 2nd
messenger signaling systems.
Cell-type Specific Factors
• Receptor Splice Variants
• Levels of receptor expression and signal amplification (see
next slide).
- with high receptor density + strong coupling to G protein
pathway, the [drug] required to generate 2nd messengers may
<< [drug] required to occupy a significant fraction of
receptors.
- This system will show a large amount of signal amplification.
- ‘receptor reserve’ = ‘spare receptors’ = ‘strong coupling’.
- Signal amplification is fast.
- Equilibrium is reached quickly – depends on the rate
constants for association and dissociation.
Signal Amplification and Receptor Reserve
Response
100
80
Response
60
% of
Max
Binding
40
20
KD = 100 nM – good
enough in a strongly
coupled system (left shift).
In contrast, the same
receptors in this cell may
also signal through another,
less well coupled pathway
with less signal amplificatio
and less receptor reserve.
10
0.01
0.1
1
10
100
Drug (nM)
1000
10000
Specificity of receptors for G protein subtypes
Some receptors can show selectivity for a certain α
subtype in 1 type of cell, but not in another cell type:
R (muscarinic)
R (somatostatin)
G (α01/β3/γ)
G (α02/β1/γ)
E (Ca2+ channels)
Restricted localization
GPCRs undergo the same trafficking we have
discussed earlier (Protein trafficking and LGIC
slides).
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.
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).
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 (Nethylmaleimide 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.
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.
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 agonistinduced 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.
Mechanisms of Receptor Regulation
D
D
D
P P
P P
α
α
(2) Phosphorylation
β
(3) Arrestin
αγ
(7) Recycling binding
(1) Agonist binding
and G protein
activation
(8) Traffic to
lysosomes
Lysosomes
P P
Arrestin
Arrestin
(4) Clustering in
clathrin-coated
pits
Clathrin
(5) Endocytosis
Endosomes D
(6) Dissociation of agonist:
• Dephosphorylation
• Sorting between cycling
and lysosomal pathways
D
P P
Arrestin
Expanding roles for
β-arrestins as
scaffolds and
adaptors in GPCR
signaling and
trafficking. (Miller &
Lefkowitz, 2001. Curr. Opin.
Cell Biol. 13:139-145).
Another Receptor – G Protein Cycle
How G-protein-coupled receptors work (1)
extracellular space
N
‘7TM’ - receptor
cytosol
g b a
GDP
Ligand
g b
a
GTP
GDP
heterotrimeric
G-protein
How G-protein-coupled receptors work (2)
N
active
a
g b
GTP
P
N
inactive
g b a
GDP
How G-protein-coupled receptors work (3)
Adenylate cyclase
active
inactive
a
GTP
g b
ATP
cAMP
Protein kinase A
cAMP
active
inactive
Phosphorylation of multiple target proteins
Some G-proteins are inhibitory
b-Adrenoceptor
AC
active
as
GTP
a2-Adrenoceptor
AC
inactive
ai
GTP
bg-Subunits of G proteins may have
regulatory activity, too
Muscarinic (M2)
acetylcholine receptor
Kir
AC
inactive
g b
K+
ai
GTP
Ga-proteins regulate diverse effector systems
as
adenylate cyclase 
cAMP 
protein kinase A 
ai
adenylate cyclase 
cAMP 
protein kinase A 
1
a
t
cGMP phosphodiesterase 
aq
phospholipase C 
PIP2
IP3 + DAG
Ca++
ER
cGMP 
protein kinase C 
phosphorylation of
multiple proteins
Many transmitters have multiple GPCR with
different downstream signaling mechanisms
Norepinephrine,
epinephrine
a1
a2
b1 , b2
IP3 + DAG
cAMP
cAMP



Dopamine
D2 - D4
D1, D5
cAMP
cAMP


Acetylcholine
M1,, M4, M5
M2 , M 3
IP3 + DAG
cAMP


Bivalent muscarinergic agonists
S
N
N
N
O
O
N
n
S
N
O
N
Dimeric drugs might target
heterooligomeric GPCR
H3C
S
NH2
H3C
F3C
O
PD 81,723
Cycloexhyladeonsine
binding (relative)
‘Allosteric’ agonists
log [PD 81,723]
Cooperative binding to GPCR oligomers
may explain the behaviour of pseudo-allosteric
agonists
Inositol-P (% basal)
Arachidonic acid (% basal)
Agonist-specific coupling of a1-adrenergic receptors
Log [agonist] (M)
Efficacy: NA = pOCT > mOCT
NA > mOCT > pOCT
Coupling to multiple G-proteins
in the two-state model
Antagonist
Agonist
GPCR
inactive
G-protein A,
Effect A
GPCR
active
G-protein B,
Effect B
Agonist-specific coupling implies multiple
active states of a GPCR
Antagonist
Agonist A
Agonist B
GPCR
inactive
G-protein A,
Effect A
G-protein B,
Effect B