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

(contd)
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 2-Adrenergic G ProteinCoupled Receptor

Vadim Cherezov, Daniel M. Rosenbaum, Michael A. Hanson, Sren 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 nucleotidebinding 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 2-adrenergic receptorT4 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 proteincoupled 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 2-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

, , Subunits
Subunit (23 isoforms): contains the GTP/GDP binding site
is responsible for identity.
(5 isoforms) and (12 isoforms): are identical or very
similar, interchangeable in vitro; most of them are
ubiquitously expressed; membrane anchored through
prenylation of G.
Golf: expressed in olfactory bulb, coupled to PLC.
GT (transducin): is coupled to cGMP phosphodiesterase and is
expressed in the rod cells of the retina (these cells are
Inactivated by light!!): h 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
5GMP (inactive => channels closed => membrane
polarization => no nt released).

Receptor Family 1 Rhodopsin Family

D
R
Y

TM7

Extracellular
TM6

C
TM5

TN4

TM3

TM2

TM1

Y Y

Lipid Bilayer
CC
Intracellular
C

Receptor-Ligand Interactions:
Rhodopsin-like Family
6
N HO
F
+N

1
3
2

O S
H S
OH

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 Bilayer
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

Drug binding
and G protein
activation

GDP

Reformation of
receptor G protein
complex

The G Protein Cycle

D
Dissociation of
receptor-G protein
complex
GTP

GTP
Inactivation of
G through intrinsic
GTPase activity

Pi

GDP

Receptor-G protein Interactions

How are receptor-G protein interactions measured?
Ligand-binding assays:

Low- affinity

High-affinity

R + G(GTP--S)

RG(GDP)
GDP

GTPS

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 2 nd 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
0.010.1 1

10 100 1000
Drug (nM)

10000

Specificity of receptors for G protein subtypes

Some receptors can show selectivity for a certain

subtype in 1 type of cell, but not in another cell type:
R (muscarinic)

R (somatostatin)

G (01/3/)
G (02/1/)

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
-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
G subunit.
Can also attenuate G protein actions that are mediated
by subunits, because they can alter the number of
available by enhancing the affinity of G subunits
for the after GTP hydrolysis incr rate of
reformation of the heterotimer.

Regulation at the G protein level (contd)

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

(2) Phosphorylation

(3) Arrestin

(7) Recycling binding

(1) Agonist binding

and G protein
activation

P P

P P

Arrestin

Arrestin

(4) Clustering in
clathrin-coated
pits

(5) Endocytosis

Endosomes D

(6) Dissociation of agonist:

(8) Traffic to () Dephosphorylation
lysosomes () Sorting between cycling
Lysosomes
and lysosomal pathways

Clathrin

P P
Arrestin

Expanding roles for

-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

GDP

Ligand

GTP
GDP

heterotrimeric
G-protein

How G-protein-coupled receptors work (2)

N

active

GTP

P
N
inactive

GDP

How G-protein-coupled receptors work (3)

Adenylate cyclase
active

inactive

GTP

ATP
cAMP
Protein kinase A

cAMP
active

inactive

Phosphorylation of multiple target proteins

Some G-proteins are inhibitory

-Adrenoceptor
AC
active

GTP

2-Adrenoceptor
AC
inactive

i
GTP

-Subunits of G proteins may have

regulatory activity, too
Muscarinic (M2)
acetylcholine receptor
Kir
AC
inactive

K+

i
GTP

G-proteins regulate diverse effector systems

s

adenylate cyclase

cAMP

protein kinase A

i1

adenylate cyclase

cAMP

protein kinase A

cGMP phosphodiesterase

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,
1
IP3 + DAG
epinephrine
2
cAMP

1 , 2
cAMP

Dopamine

D2 - D4
cAMP

D1, D5
cAMP

Acetylcholine

2, M3
cAMP

IP3 + DAG

Bivalent muscarinergic agonists

S
N

N
O

S
N

Dimeric drugs might target

heterooligomeric GPCR

H3C

S
NH2

H3C

F3C

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 1-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