Nature Reviews Molecular Cell Biology 3, 639-650 (2002)

1
G-Protein Coupled Receptors (GPCRs)Lectures
February 28, March 2, 7, 9 and 11, 2005 Michael
Greenwood (michael.greenwood_at_mcgill.ca)
Nature Reviews Molecular Cell Biology 3, 639-650
(2002) SEVEN-TRANSMEMBRANE RECEPTORS

Kristen L. Pierce, Richard T. Premont
Robert J. Lefkowitz The Howard Hughes Medical
Institute and the Departments of Medicine and
Biochemistry, Box 3821, Duke University Medical
Center, Durham, North Carolina, 27710,
USA. Seven-transmembrane receptors, which
constitute the largest, most ubiquitous and most
versatile family of membrane receptors, are also
the most common target of therapeutic drugs.
Recent findings indicate that the classical
models of G-protein coupling and activation of
second-messenger-generating enzymes do not fully
explain their remarkably diverse biological
actions.
This is the main review for the GPCR
lectures. Most advanced textbooks (i.e.Mol. Biol.
of the Cell) cover the basics of GPCRs
2
Lecture topicsA. GPCRs as receptors 1-Basic
structure -7 transmembrane topology -receptors
are associated with a heterotrimeric G-protein 2-
Diversity of the GPCR gene family -3
subfamilies -GPCRs mediate the effects of a
large variety of different agonists -multiple
receptors recognize the same ligand 3- Receptor
activation of heterotrimeric G-proteins -model
of GPCR activation -different G-proteins
activate distinct signalling pathways -receptor
specificity -diversity of GPCR
signalling -G-protein independent GPCR
signalling 4- Functional domains on the
GPCR -receptor pharmacology -G-protein
activating domains -ligand binding pocket or
domains 5- Inactivation of GPCR mediated
signalling -tachyphylaxis -receptor
desensitization at the molecular level GRKs and
arrestins -inactivation of the receptor
activated heterotrimeric G-protein by RGSs 6-
GPCR dimerization 7- Alternative function of
GPCRs B. Topics in GPCR biology (if time
permits) 1-Odorant receptors 2-GPCRs as drug
targets orphan and known GPCRs 3-Agonist
independent activation of Angiotensin II
receptors in the heart by stretch 4-Chemokine
receptors cancer metastasis molecular piracy by
virally encoded GPCRs as entry points for HIV
and malaria
3
1. Basic structure - 7 Transmembrane Domains
(TMDs), 3 intracellular loops, 3 extracellular
loops, N- and C-teminals
Fig. 1. Schematic representation of the membrane
topology of the human ß 2 adrenergic receptor.
The localizations of TMHs in the human
ß 2-adrenoceptor are indicated (black lines). The
core and water-lipid interface regions of the
lipid membrane are indicated with light gray and
dark gray colors on the background.
Each of the 7 TMHs have one characteristic
residue (black circles with white text), which is
found among the majority of family 1 (also called
A) GPCRs.
Pharmacol Ther. 2004 Jul103(1)21-80
4
1. Basic structure
Cysteine bridges
Two-dimensional topology of the human CCR5
sequence. Membrane topology of CCR5 with the
extracellular space at the top and the
intracellular space at the bottom. Amino acids
shown to be critical for CCR5 function are
highlighted by filled circles. The grey box marks
the approximate position of the membrane bilayer.
Cell Signal. 2004 Nov16(11)1201-10
5
1. Basic structure GPCRs are lipoproteins
Schematic representation of a family A receptor
in the cell membrane based on the packing
arrangement of TMHs observed in the most recent
crystal structure of rhodopsin (pdb code 1L9H).
Putative TMHs are depicted as cylinders.
Pharmacol Ther. 2004 Jul103(1)21-80
6


1. Basic Structure Coupled to heterotrimeric
G-protein

(A) GPCRs have a central common core made of
seven transmembrane helices (TM-I to -VII)
connected by three intracellular (i1, i2, i3) and
three extracellular (e1, e2, e3) loops. (B)
Illustration of the central core of rhodopsin.
The core is viewed from the cytoplasm. The length
and orientation of the TMs are deduced from the
two-dimensional crystal of bovine and frog
rhodopsin (Unger et al., 1997).
The EMBO Journal (1999) 18, 17231729
7
Classification and diversity of GPCRs. (A) Three
main families (1, 2 and 3) can be easily
recognized when comparing their amino-acid
sequences. Receptors from different families
share no sequence similarity, suggesting that we
are in the presence of a remarkable example of
molecular convergence. Family 1 contains most
GPCRs including receptors for odorants. Group 1a
contains GPCRs for small ligands including
rhodopsin and ß-adrenergic receptors. The binding
site is localized within the seven TMs. Group 1b
contains receptors for peptides whose binding
site includes the N-terminal, the extracellular
loops and the superior parts of TMs. Group 1c
contains GPCRs for glycoprotein hormones. It is
characterized by a large extracellular domain and
a binding site which is mostly extracellular but
at least with contact with extracellular loops e1
and e3. Family 2 GPCRs have a similar morphology
to group Ic GPCRs, but they do not share any
sequence homology. Their ligands include high
molecular weight hormones such as glucagon,
secretine, VIP-PACAP and the Black widow spider
toxin, a-latrotoxin. Family 3 contains mGluRs and
the Ca2 sensing receptors. Last year, however,
GABA-B receptor and a group of putative pheromone
receptors coupled to the G protein Go (termed VRs
and Go-VN) became new members of this family. (B)
Family 4 comprises pheromone receptors (VNs)
associated with Gi. Family 5 includes the
'frizzled' and the 'smoothened' (Smo) receptors
involved in embryonic development and in
particular in cell polarity and segmentation.
Finally, the cAMP receptors (cAR) have only seen
found in D.discoïdeum but its possible expression
in vertebrate has not yet been reported.
2. Diversity of the GPCR superfamily
The EMBO Journal (1999) 18, 17231729
8
2. Diversity. -large variety of different
agonists
9
2. Diversity (of physiological responses to GPCR
stimulation)
TARGET TISSUE HORMONE MAJOR RESPONSE
Thyroid gland thyroid-stimulating hormone (TSH) thyroid hormone synthesis and secretion
Adrenal cortex adrenocorticotrophic hormone (ACTH) cortisol secretion
Ovary luteinizing hormone (LH) progesterone secretion
Muscle adrenaline glycogen breakdown
Bone parathormone bone resorption
Heart adrenaline increase in heart rate and force of contraction
Liver glucagon glycogen breakdown
Kidney vasopressin water resorption
Fat adrenaline, ACTH, glucagon, TSH triglyceride breakdown

TARGET TISSUE SIGNALING MOLECULE MAJOR RESPONSE
Liver vasopressin glycogen breakdown
Pancreas acetylcholine amylase secretion
Smooth muscle acetylcholine contraction
Blood platelets thrombin aggregation

10
2. Diversity Multiple GPCRs can bind a single
agonist serotonin.
Serotonin (5-hydroxytryptamine or 5-HT) is
involved in mediating a large number of different
responses and diseases. These are now seven
sub-families of 5HT receptors, 5-HT17,
comprising a total of 14 structurally and
pharmacologically distinct mammalian 5-HT
receptor subtypes.
Fig. 1. Dendrogram showing the evolutionary
relationship between various human 5-HT receptor
protein sequences (except 5-HT5A and 5-HT5B
receptors which are murine in origin).
11
2. Diversity.
Fig. 1. Graphical representation of the current
classification of 5-HT receptors. Receptor
subtypes represented by coloured boxes and lower
case designate receptors that have not been
demonstrated to definitively function in native
systems. Abbreviations 3'-5' cyclic adenosine
monophosphate (cAMP) phospholipase C (PLC)
negative (-ve) positive (ve).
Pharmacol Biochem Behav. 2002 Apr71(4)533-54
12
Receptor subtype Ligands with highest affinity

Cholecystokinin
    CCK1 Sulfated CCK
    CCK2 Sulfated CCK, nonsulfated CCK, gastrin
Endothelin
    ETR-A Endothelin-1
    ETR-B Endothelin-1, -2, -3
NPY
    Y1 NPY, PYY
    Y2 NPY, NPY(336), PYY(336)
    Y4 PP
    Y5 NPY, PYY
Orexin
    Orexin A/hcrt1 Orexin A
    Orexin B/hcrt2 Orexin A, orexin B
Somatostatin
    sstR1-sstR4 sst14, Cortistatin-14, -29
    sstR5 sst28, Cortistatin-14, -29
2. Diversity GPCR subfamilies multiple
receptors often recognize the same ligand
TABLE 2. Examples of specificity and multiplicity
of peptide ligand-receptor interactions
Endocrinology Vol. 145, No. 6 2645-2652

13
3. Receptor activation of heterotrimeric
G-proteins. Basic model
Hollinger et al. 2000 Pharmacological Reviews
14
3. Receptor activation Simplified Model of GPCR
activation
A schematic representation of how the two-state
receptor model relates to the action of drugs as
strong agonists, partial agonists, neutral
competitive antagonists, inverse agonists, and
inverse partial agonists. The inactive and active
receptor conformations (R and R, respectively)
are in constant equilibrium. A strong agonist
binds selectively to R, driving the equilibrium
between R and R in favour of R, resulting in
enhanced response. A partial agonist has higher
affinity for R than for R, but with less
selectivity than the strong agonist. The neutral
competitive antagonist binds with equal affinity
to both R and R, so that it does not disturb the
resting equilibrium and therefore does not alter
basal response. An inverse strong agonist binds
selectively to R, driving the equilibrium between
R and R in favour of R, resulting in decreased
response, that is, when there is significant
constitutive activity (basal response). An
inverse partial agonist has higher affinity for R
than for R, but with less selectivity than the
strong inverse agonist        
British Journal of Clinical Pharmacology 
57 (4), 373-387.
15
3. Receptor activation Allosteric model of GPCR
activation
????????????????????
Dissecting the allosteric two-state model. The
allosteric two-state model cube.
16
3. Receptor activation GPCRs activate
different sub-classes of heterotrimeric
G-proteins and effector systems
17
3. Receptor activation GPCRs activate different
sub-classes of heterotrimeric G-proteins and
effector systems (contd)
Nature Reviews Molecular Cell Biology 3 639-650
18
3. Receptor activation Activation of cAMP
responses by Gs coupled GPCRs
How gene transcription is activated by a rise in
cyclic AMP concentration.
Molecular Biology of the Cell
19
3. Receptor activation Activation of
phospholipase Cß by Gq coupled GPCRs
The hydrolysis of PI(4,5)P2 by phospholipase C-b.
Molecular Biology of the Cell
20
3. Receptor activation
Receptor switching
Possible mechanism underlying the "switch" of the
functional coupling of a given receptor with
distinct G-proteins. Stimulation of the naïve
receptor favours the coupling with a subset of
G-proteins, resulting in the activation of a
preferential signalling cascade (Response A).
This response includes the activation of a
protein kinase that may phosphorylate the
receptor and thereby progressively impair the
coupling with this subset of G-proteins. In
contrast, while response A is progressively
inhibited, the coupling of the phosphorylated
receptor with another subset of G-proteins is
maintained or even enhanced, leading to the
emergence of another signalling cascade (Response
B).
Pharmacol Ther. 2003 Jul99(1)25-44
21


3. Receptor activation GPCRs are unfaithful to
G proteins

How can such interactions be characterized?
and/or identified????
Two examples of transduction triggered via a
direct interaction of GPCRs with proteins
containing PDZ and EVH-like domains.
22
3. Receptor activation Receptor independent
activation of heterotrimeric G-Proteins
? ?
AGS
? ?
GPCRs
Signal regulator that influences the transfer of
signal from receptor to G-protein or directly
regulates the activation state of G-proteins.
Biol Cell. 2004 Jun96(5)369-72.
23
3. Receptor activation Multiple receptors
activate the same G-protein
24
3. Receptor activation Complexity of GPCR
signalling Cascades
GPCRs cross talk with Receptor Tyrosine Kinases
(RTK)
Given such a diversity in responses, how does
GPCR signaling specificity occur???
Multiple physiological responses
25
3. Receptor activationYeast as a model system
to study GPCR structure, function and receptor
specificity
Saccharomyces cerevisiae
Budding yeast Bakers yeast Brewers yeast
26
3. Receptor activation Receptor
Specificity Yeast has 2 distinct GPCR signalling
cascades
Versele et al. 2001
27
3. Receptor activation Signalling Specificity
is achieved by Scaffolding in Yeast
Cartoon of Ste5p and Far1p scaffolds. Ste5p is
required for activation of the mating MAPK
cascade in response to mating pheromone and does
not have an intrinsic kinase activity. Far1p is
required for oriented polarized growth in
response to mating pheromone. Far1p is postulated
to be an analog of Ste5p on the basis of its
ability to associate with multiple components of
an individual signal transduction pathway, but it
is not known whether they simultaneously bind to
associated signaling components.
28
3. Receptor activation In mammalian cells, GPCR
specificity is illustrated by GPCR mediated
activation of MAPK cascades
7TM receptors activate the ERK/MAPK cascade by
several different pathways 
Nature Reviews Molecular Cell Biology 3 639-650
(2002)
29
3. Receptor activation Scaffolding of MAPK
cascade is also seen in mammalian cells
Nature Reviews Molecular Cell Biology 3 639-650
(2002)
30
3. Receptor activation Microdomains can also
contribute to GPCR specificity Caveolae
Schematic representation of the lipid and protein
organization of a caveola. Sphingolipid- and
cholesterol-rich domain is shown in red and
nonraft lipid domains are shown in blue. Caveolae
contain a coat of oligomeric caveolin molecules
inserted into the cytoplasmic leaflet of the
membrane. Some proteins, including certain GPCR
(shown as heptahelical structures with associated
G protein), partition to caveolar domains due to
either acylation, binding to caveolin or
formation of a sphingolipid shell around the
protein (or by a combination of these, and/or yet
unknown, mechanisms). Also shown are undefined
cytoskeletal interacting proteins (orange, green,
purple) and noncaveolar membrane proteins (blue)
and partners (light blue).
31
3. Receptor activation Diversity multiple GPCRs
are expressed in the same cell/tissue
Example of blood vessels- molecular biology is
trying to understand the complexity of GPCR
responses seen in in vivo situations.
32
3. Receptor activation Diversity
Example of cardiac cells.
British Journal of Anaesthesia, 2004, 9334-52
Sympathetic and parasympathetic signalling
cascades of G-protein coupled receptors down to
the level of cellular responses. Note the
intimate crosstalk between the various signalling
pathways. Lines with blunted ends () indicate
inhibition. ACadenylyl cyclase
AChacetylcholine ARadrenergic receptor
cAMPcyclic AMP cGMPcyclic GMP
DAGdiacylglycerol ET1endothelin receptor-1
GCguanylyl cyclase G  i, G  s, G  q, Gß
 G-protein subunits IP3inositol trisphosphate
M2muscarinic acetylcholine receptor
MAPKmitogen activated protein kinase NOSnitric
oxide synthase PDK1phosphoinositide-dependent
kinase-1 PI3Kphosphoinositide-3 kinase PKA,
PKB, PKC, PKGtarget-specific serinethreonine
protein kinases PLCphospholipase C Rassmall
monomeric GTPase RNOSreactive nitric oxide
species.