Signal Reception: G Protein-Coupled 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 Subunitsα 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
TM2
TM3
TM1
TN4
TM5
TM6
TM7
NY Y
Extracellular
IntracellularDRY
CC
C
C C
Lipid Bilayer
Receptor-Ligand Interactions:Rhodopsin-like Family
32
1
7
6
4
5N
F
D
+N
HO
OHOH
S
S
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
TM2
TM3
TM1
TN4
TM5
TM6
TM7
Y Y
Extracellular
IntracellularDRY
CC
C
C C
Lipid Bilayer
N
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 D
ααβ
αγ
GDP
Drug bindingand G protein
activationααβ
αγ
GTP
D
Dissociation ofreceptor-G protein
complex
αα
βα
γ GTP
Pi
αα
GDP
Reformation ofreceptor G protein
complex
Inactivation of Gα through intrinsic
GTPase activity
The G Protein Cycle
Receptor-G protein InteractionsHow are receptor-G protein interactions measured?
• Ligand-binding assays:
High-affinity Low- affinityRG(GDP)
GDP GTPγS
R + G(GTP-δ-S)
Without GTP, both high- and low-affinity states are measured.With GTP and Mg2+, only low-affinity state is measured, becauseAgonist 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.
% ofMax
100
80
60
40
20
10
Binding
Response
Response
0.01 0.1 1 10 100 1000 10000Drug (nM)
Signal Amplification and Receptor Reserve
KD = 100 nM – goodenough in a stronglycoupled system (left shift).In contrast, the same receptors in this cell may also signal through another, less well coupled pathway with less signal amplification and less receptor reserve.
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) E (Ca2+ channels)
G (α02/β1/γ)
Restricted localizationGPCRs 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.
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.
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.
D D D D
αα
βα γ
(1) Agonist bindingand G protein
activation
(2) PhosphorylationP 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
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
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
aGTP
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
asGTP
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
as adenylate cyclase protein kinase A cAMP
ai1 adenylate cyclase protein kinase A cAMP
aq phospholipase C
PIP2 IP3 + DAG protein kinase C
phosphorylation ofmultiple proteins
Ca++
ER
at cGMP phosphodiesterase cGMP
Many transmitters have multiple GPCR with different downstream signaling mechanisms
Norepinephrine, a1 IP3 + DAG epinephrine a2 cAMP
b1, b2 cAMP
Dopamine D2 - D4 cAMP D1, D5 cAMP
Acetylcholine M1,, M4, M5 IP3 + DAG M2, M3 cAMP
Bivalent muscarinergic agonists
N
NS
NO
OO
N
N S
Nn
Dimeric drugs might target heterooligomeric GPCR
S
OF3C
CH3
CH3
NH2
PD 81,723 log [PD 81,723]
Cyc
loex
hyla
deon
sine
bi
ndin
g (r
elat
ive)
‘Allosteric’ agonists
Cooperative binding to GPCR oligomers may explain the behaviour of pseudo-allosteric
agonists
Agonist-specific coupling of a1-adrenergic receptors
Ara
chid
onic
aci
d (%
bas
al)
Inos
itol-P
(% b
asal
)
Log [agonist] (M)
Efficacy: NA = pOCT > mOCT NA > mOCT > pOCT
Coupling to multiple G-proteins in the two-state model
GPCRactive
Agonist
GPCRinactive
Antagonist
G-protein A,Effect A
G-protein B,Effect B
Agonist-specific coupling implies multiple active states of a GPCR
Agonist B
GPCRinactive
G-protein A,Effect A
G-protein B,Effect B
Agonist AAntagonist