G-Protein Coupled Receptors (GPCRs)
Lectures: February 28, March 2, 7, 9 and 11, 2005;
Michael Greenwood (michael.greenwood@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
Lecture topics:
A. 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
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 β 2 adrenergic
receptor. The localizations
of TMHs in the human β 2adrenoceptor 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 Jul;103(1):21-80
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 Nov;16(11):1201-10
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 Jul;103(1):21-80
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 twodimensional crystal of bovine and
frog rhodopsin (Unger et al.,
1997).
The EMBO Journal (1999) 18, 1723–1729
2. Diversity of the GPCR superfamily
The EMBO Journal (1999) 18, 1723–1729
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 βadrenergic 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, α-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….
-large variety of different agonists
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
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-HT1–7, 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 5HT5B receptors which are murine in origin).
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 Apr;71(4):533-54
Receptor
subtype
Ligands with highest affinity
Cholecystoki
nin
CCK1
Sulfated CCK
CCK2
Sulfated CCK, nonsulfated
CCK, gastrin
2. Diversity…
GPCR subfamilies: multiple
receptors often recognize the
same ligand
Endothelin
ETR-A
Endothelin-1
ETR-B
Endothelin-1, -2, -3
NPY
Y1
NPY, PYY
Y2
NPY, NPY(3–36), PYY(3–36)
Y4
PP
Y5
NPY, PYY
TABLE 2. Examples of specificity
and multiplicity of peptide ligandreceptor interactions
Orexin
Orexin
A/hcrt1
Orexin A
Orexin
B/hcrt2
Orexin A, orexin B
Somatostatin
sstR1sstR4
sstR5
sst14, Cortistatin-14, -29
sst28, Cortistatin-14, -29
Endocrinology Vol. 145, No. 6 2645-2652
3. Receptor activation of heterotrimeric G-proteins.
Basic model
Hollinger et al. 2000 Pharmacological Reviews
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.
3. Receptor activation
Allosteric model of
GPCR activation
????????????????????
Dissecting the allosteric two-state model. The allosteric two-state model cube.
3. Receptor activation…
GPCRs activate different
sub-classes of heterotrimeric
G-proteins and effector systems
3. Receptor activation…
GPCRs activate different sub-classes of heterotrimeric
G-proteins and effector systems (cont’d)
Nature Reviews Molecular Cell Biology 3; 639-650
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
3. Receptor activation…
Activation of phospholipase Cβ by Gq coupled GPCRs
The hydrolysis of PI(4,5)P2 by phospholipase C-b.
Molecular Biology of the Cell
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 Gproteins, 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 Jul;99(1):25-44
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.
3. Receptor activation…
Receptor independent activation of heterotrimeric G-Proteins
GPCRs →
→
←
←
AGS
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 Jun;96(5):369-72.
3. Receptor activation…
Multiple receptors activate the same G-protein
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
3. Receptor activation…
Yeast as a model system to study GPCR structure, function and receptor
specificity
Saccharomyces cerevisiae
Budding yeast
Baker’s yeast
Brewer’s yeast
3. Receptor activation…
Receptor Specificity:
Yeast has 2 distinct GPCR signalling cascades
Versele et al. 2001
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.
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)
3. Receptor activation…
Scaffolding of MAPK cascade is also seen in mammalian cells
Nature Reviews Molecular Cell Biology 3; 639-650 (2002)
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).
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.
3. Receptor activation…
Diversity…
Example of cardiac cells.
British Journal of Anaesthesia, 2004, 93;34-52
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. AC=adenylyl cyclase; ACh=acetylcholine;
AR=adrenergic receptor; cAMP=cyclic AMP; cGMP=cyclic GMP; DAG=diacylglycerol; ET1=endothelin
receptor-1; GC=guanylyl cyclase; G i, G s, G q, Gß =G-protein subunits; IP3=inositol trisphosphate;
M2=muscarinic acetylcholine receptor; MAPK=mitogen activated protein kinase; NOS=nitric oxide
synthase; PDK1=phosphoinositide-dependent kinase-1; PI3K=phosphoinositide-3 kinase; PKA, PKB, PKC,
PKG=target-specific serine–threonine protein kinases; PLC=phospholipase C; Ras=small monomeric
GTPase; RNOS=reactive nitric oxide species.