3. Receptor activation…
Diversity…
Models of the
Integration of
complex GPCR
signalling
networks
Evolution of the complexity of our perception of GPCR-mediated signaling from a pathway
(A) to matrices (B) to networks (C). Receptors (R) interact with one or more G proteins (G) linked
to one or more effectors (E), which then engage in effector cross-talk and regulation of cellular
function at the surface membrane, within the cytosol, and among multiple cellular compartments,
including the nucleus.
Molecular Interventions 4:326-336, (2004)
4. Functional regions within GPCRs:
4.1 G-protein interacting domains
4.2 ligand binding domain
The three subfamilies of
GPCRs are depicted with
examples of their
endogenous agonists.
The binding modes of the
orthosteric ligands for
each receptor type are
depicted by a green
rectangle. The GPCR
signals either by coupling
to heterotrimeric Gproteins consisting
of and
subunits
(which trigger a wide
range of metabolic
cascades and ion
channel activities) or by
direct association with
effector molecules. AC,
adenylyl cyclase; ATP,
adenosine triphosphate;
cAMP, cyclic adenosine
monophosphate; PLC,
phospholipase C; IP3,
inositol-3,4,5-trisphosphate; DAG,
diacylglycerol.
4.Functional regions within GPCRs
4.1 G-protein coupling domain
Using GPCR chimeras to identify
Functional domains within receptors
Two different dopamine
receptors bind same
agonist but they couple to
different signalling
cascade
Mol Pharmacol. 2004 65:1323-32.
4. Functional regions within GPCRs
Pharmacological characterization of GPCRs
(A) Saturation isotherms:
Determine the number of high affinity
binding sites on the cell surface
(B) Displacement analysis:
Determine the affinity of the GPCR for
different agonists and antagonists
Journal of Neuroendocrinology 16, 356-361
Binding properties of tritiated d[Cha4]AVP. Membranes from
COS cells, expressing human V1b receptors (1-2 µg
protein/assay), were incubated for 1 h at 37 °C with
increasing amounts of tritiated d[Cha4]AVP (a) or with 1-2 nm
of the radioligand with, or without (control), increasing
amounts of unlabelled selective vasopressin antagonists
(SR149415, SR49059, SR121463) (b).
4.1 G-protein coupling domain…
Analysis of vasopressin
receptor chimeras
Structure, ligand binding properties, and functional profile of wild type and mutant V1 /V2
vasopressin receptors. [ H]AVP saturation binding studies were carried out. Km and Bmax
values are given as means ± S.E. of three independent experiments (PI, stimulation of PI
hydrolysis; AC, stimulation of adenylyl cyclase). The symbols are defined as the percentage of
maximum PI and cAMP responses induced by the wild type V1a and V2 receptor, respectively:
++++, 90-100%; +++, 80-90%; +, 10-30%; -, no significant response.
J. Biol. Chem. 1996;271:8772
4.1 G-protein coupling domain…
Analysis of the potency of
Vasopressin chimeras to stimulate
cAMP accumulation (Gs response)
AVP-induced cAMP accumulation mediated by wild type V1a, V2 and hybrid
V1a/V2 vasopressin receptors. Transfected COS-7 cells transiently expressing the
different receptors were incubated in 6-well plates for 1 h at 37 °C with the indicated
AVP concentrations, and the resulting increases in intracellular cAMP levels were
determined. The data are presented as fold increase in cAMP above basal levels in
the absence of AVP. Each curve is representative of three independent experiments,
each carried out in duplicate.
J. Biol. Chem. 1996;271:8772
4.1 G-protein coupling domain…
Analysis of the potency of
Vasopressin chimeras to stimulate
Inositol-Phosphate (PI or lipid
hydrolysis) accumulation
(Gq response)
Figure 3: AVP-induced stimulation of PI hydrolysis mediated by wild type V1a, V2
and hybrid V1a/V2 vasopressin receptors. Transfected COS-7 cells transiently
expressing the various receptors were incubated in 6-well plates for 1 h at 37 °C
with the indicated AVP concentrations, and the resulting increases in intracellular IP
levels were determined. The data are presented as fold increase in IP above basal
levels in the absence of AVP. Each curve is representative of three independent
experiments, each carried out in duplicate.
J. Biol. Chem. 1996;271:8772
4.1 G-protein coupling domain…
Fine structure mapping of
G-protein interacting region
using deletion analysis
Localization of FSHR Mutations and Functional Characterization of Mutant Receptors
To characterize mutant FSHRs (A), COS-7 cells were transfected with the different
constructs, and cAMP accumulation assays were performed as outlined in Materials and
Methods. (B) Data obtained from three independent experiments, each performed in
triplicate, are presented as -fold of basal cAMP levels of the FSHR(wt) (means ± SEM).
Molecular Endocrinology 1999, 13: 181-190
4.1 G-protein coupling domain…
Fine structure mapping of
G-protein interacting region
using alanine mutagenesis
Functional Analysis of Conserved Basic Amino Acids within the LHR i2 Loop by Alanine
Scanning Mutagenesis. To identify a cationic contact site for D564, all conserved basic
amino acids within the i2 loop were replaced by A using a site-directed mutagenesis
approach (A). The various LHR mutants were expressed in COS-7 cells, and cAMP
accumulation assays were performed (B). Basal (open bars) and agonist-induced cAMP
levels are presented as means ± SEM of two independent experiments, each carried out
in triplicate.
Molecular Endocrinology 1999, 13: 181-190
4. Functional regions within GPCRs
4.2 Ligand binding domain
3-D models
Schematic models of ligand-receptor complexes for structurally diverse ligands
interacting with GPCRs.
4. Functional regions within GPCRs
4.2 Ligand binding domain
Using GPCR chimeras to identify
Functional domains within receptors
Two different receptors one
binds FSH and the other binds
LH
Mol Pharmacol. 2004 Jun;65(6):1323-32.
4.2 Ligand binding domain…
Structure of Cholecystokinin R eceptor Binding Sites and Mechanism of
Activation/Inactivation by Agonists/Antagonists
A wide repertoire of physiological effects of CCK and/or gastrin which mediated CCK1 and/or CCK2 receptors
has been identified. Among actions of CCK which are mediated by the CCK1R, control of satiety, gallbladder
contraction, pancreatic exocrine secretion, gastric pepsinogen and leptin secretions, gastric emptying and gut
motility are the best known. Actions of CCK which occur through the CCK2R include modulation of anxiety and
pain perception; these actions involve CCK2 receptors of the central nervous system. The wide spectrum of
biological functions regulated by the CCK1R and CCK2R makes them candidate targets for a therapeutic
approach in a number of diseases. This led a number of academic and pharmaceutical research groups to
design specific and highly potent agonists and antagonists for those receptors.
As for other G-protein-coupled receptors, the cloning of CCK receptor cDNAs and genes have stimulated
generation of new biological models (transgenic animals, genetically modified cells ) and opened new
avenues for research in the physiology, pathophysiology, molecular pharmacology and structure-function
relationships of the receptors. Among these themes, delineation of CCK receptor binding sites represents a
prerequisite for the understanding of the molecular basis for ligand recognition, partial agonism, ligandinduced traffiking of receptor signalling etc.
Pharmacology & Toxicology 2002 91;313
4.2 Ligand binding domain…
Criteria used to identify residues of CCKR binding site in an example of analysis
of a putative ionic interaction between a positively charged aminoacid Y+ of the
receptor binding site, and its negatively charged partner residues X in the ligand.
Pharmacology & Toxicology 91:313, 2002
Schematic representations of the CCK1R with residues of the CCK binding site.
Pharmacology & Toxicology 91:313, 2002
4.2 Ligand binding domain…
Detailed understanding of
residues involved in ligand
binding allows detailed ligand/
Receptor modeling and permits
the application of rational drug
design to develop new agonists
and antagonist that can be used
as receptor specific drugs.
Localizations of residues in biogenic amine receptors that are important for ligand binding
(gray color) within a membrane topology model of the rat 5HT2A receptor (N terminus and most
of C terminus are not shown).
Pharmacol Ther. 2004 Jul;103(1):21-80
4. Functional regions within GPCRs
Summary
Generic diagram of sequence regions involved in post-translational
modification and modulation of functions. Summary of findings based on receptor
mutagenesis and construction of receptor chimeras for a variety of GPCR.
Molecular Interventions 4:326-336, (2004)
5. Inactivation of GPCR signalling
-Tachyphylaxis of the adrenomedulin (AM) receptor
(a) Concentration-dependent increase in cAMP in response to
AM (EC50 3.2±0.7 nM, filled squares) and CGRP at 1 µM
(no effect, filled triangle) in Rat-2 fibroblasts.
(b) Concentration-dependent attenuation of AM cAMP
responses following preexposure to AM. Rat-2 cells were
incubated with SFM or various concentrations (as indicated)
of AM for 1 h followed by a washout period and a
restimulation of 15 min with 10 nM AM Data are expressed
as a percentage of the response in cells not preincubated
with AM, that is, nondesensitised controls. Cells incubated
with AM for 1 h were less able to elevate cAMP upon
restimulation with AM when compared with cells incubated
with SFM for the same time.
(c) The time course for AM receptor desensitisation in Rat-2
cells was determined by varying the preincubation time
period (as indicated) of exposure to 100 nM AM. Cells were
then washed and restimulated, and the cAMP response was
measured. The cAMP response elicited by 10 nM AM was
attenuated in a time-dependent manner. Cells preexposed
to 100 nM AM for 1 min were capable of only 59.1±7% of
the response of cells not preincubated with AM. After 2 h
preincubation with 100 nM AM, the level of cAMP response
was 19.3±2.2% of control stimulation.
Regul Pept. 2003;112:139
5. Inactivation of GPCR signalling…
GPCR Kinase and -arrestin-dependent
desensitization and internalization of GPCRs
Fergusson, 2001
5. Inactivation of GPCR signaling…
Visulialization of GPCR internalization following receptor stimulation
Translocation of ß-arrestin 2-GFP to the ß2-adrenergic receptor (ß2AR). HEK 293
cells stably overexpressing the ß2AR were transiently transfected with ß-arrestin 2GFP. The distribution of ß-arrestin 2-GFP fluorescence was visualized by confocal
microscopy before (−Iso) and after a 5 min treatment with isoproterenol (+Iso; is a
ßAR agonist, 10−8, 10−6 M) at 37 °C. Before agonist-stimulation, ß-arrestin 2-GFP is
uniformly distributed throughout the cytosol. Upon agonist addition, ß-arrestin 2GFP translocates from the cytosol to the plasma membrane where it is found
colocalizing with the receptor in punctuated areas of the plasma membrane.
Prog Neurobiol. 2002 Feb;66(2):61-79
5. Inactivation of GPCR signaling…
Model depicting the regulation of β2AR internalization, trafficking,
dephosphorylation, and recycling
Fergusson, 2002
5. Inactivation of GPCR
signaling…
Model depicting the regulation
of GPCR internalization,
trafficking, dephosphorylation,
and recycling.
Fig. 8. Pathways involved in desensitization and resensitization of GPCR signaling. Typically
activation of a GPCR leads to (1) activation and inhibition of specific signaling pathways in the
cell, (2) short-term desensitization mediated by phosphorylation of GPCRs by GRKs followed
by β-arrestin binding to GPCRs that uncouple the receptor at the plasma membrane from the
G-protein, (3) endocytosis of the receptor, followed by postendocytic sorting of the receptor
either (4) back to the plasma membrane or (5) to lysosomes for degradation.
Molecular Interventions 4:326-336, (2004)
5. Inactivation of GPCR signaling…
Structure of GRKs
Schematic representation of the domain architecture for GRK1-GRK7. The aminoterminal GPCR-binding domain of GRK1-GRK7 contains a conserved RGS domain.
The plasma membrane targeting of each of the GRKs is mediated by distinct
mechanisms that involves their carboxyl-terminal domains. GRK1 and GRK7 are
farnesylated at CAAX motifs in their carboxyl termini. The carboxyl-terminal domains
of GRK2 and GRK3 contain a βγ-subunit binding domain that exhibits sequence
homology to a pleckstrin homology domain. The GRK5 carboxyl-terminal domain
contains a stretch of 46 basic amino acids that mediate plasma membrane
phospholipid interactions
Pharmacol Rev. 2001;53:1-24.
5. Inactivation of GPCR signaling…
Nature Reviews Molecular Cell Biology 3; 639-650 (2002)
5. Inactivation of GPCR signaling…
Regulators of G-protein Signalling (RGS) negatively regulate GPCR signalling
N
Extracellular Environment
C
GDP



GTP



+
Pi
RGS
Downstream effectors
5. Inactivation of GPCR signaling…
Prototypical RGS members
Wieland and Mittmann 2003
5. Inactivation of GPCR signaling…
Up-regulation of RGS4 desensitises endothelin-1 signalling in failing human
myocardium
In Congestive
Hearts or Sepsis
Also see
increases in
RGS1, 3, 16
Altered RGS expression leads to
changes in GPCR signalling.
Wieland and Mittmann 2003
6. GPCR dimerization:
The Split Receptor
Schematic representation of the wild-type human M2 and rat M3, the fragments M2trunc and M3-tail, and the mutants M3-short and M2 (Asn404 to Ser) muscarinic
receptors. The truncated fragment, M2-trunc, contains the amino-terminal domain,
the first five hydrophobic transmembrane regions and the initial portion (56 amino
acids) of the i3 loop of the wild-type muscarinic M2 receptor. The M3-tail fragment
contains the final portion of the i3 loop (105 amino acids), the last two hydrophobic
transmembrane regions, and the carboxy-terminal segment of the wild-type M3
muscarinic receptor. The short construct (M3-short) represents a receptor in which
196 amino acids of the i3 loop have been deleted; the remaining i3 loop is 43 amino
acids long. The point mutant M2 (Asn404 to Ser) has the asparagine 404 replaced
with serine.
6. GPCR dimerization…
Figure 1 | Role of homoand heterodimerization
in the transport of Gprotein-coupled
receptors. When
expressed alone, the
GABABR1 (GBR1) receptor
is retained as an immature
protein in the endoplasmic
reticulum (ER) of cells and
never reaches the cell
surface. By contrast, the
GBR2 isoform is
transported normally to
the plasma membrane but
is unable to bind GABA
and thus to signal. When
coexpressed, the two
receptors are properly
processed and transported
to the cell surface as a
stable dimer, where they
act as a functional
metabotropic GABAB
receptor.
Nat Rev Neurosci. 2001 Apr;2(4):274-86
6. GPCR dimerization…
Molecular determinants of G-protein-coupled-receptor dimerization. Distinct
intermolecular interactions were found to be involved for various G-protein-coupled
receptors. Covalent disulphide bonds were found to be important for the dimerization of the
calcium-sensing and metabotropic glutamate receptors. A coiled-coil interaction involving the
carboxyl tail of the GBR1 and GBR2 receptors is involved in the formation of their
heterodimer. Finally, for monoamine receptors such as the β2-adrenergic and dopamine
receptors, interactions between transmembrane helices were proposed to be involved.
6. GPCR dimerization…
Alternative three-dimensional models showing dimers of G-protein-coupled
receptors. Two models have been proposed for the general three-dimensional organization
of G-protein-coupled-receptor dimers. a | First is the domain-swapping model in which each
functional unit within the dimer is composed of the first five transmembrane domains of one
polypeptide chain and the last two of the other. Such a model is useful to rationalize the
functional complementation observed when mutant or chimeric receptors are coexpressed. b |
Second is the contact model in which each polypeptide forms a receptor unit that touches the
other through interactions involving transmembrane domains five and six.
6. GPCR dimerization…
Heterodimerization of CRLR and RAMP. HOMOTROPIC interactions between G-protein-coupled
receptors (GPCRs) are not the only type of protein–protein interaction shown to influence their
functional expression. Recently, a new class of membrane proteins that can interact with GPCRs and
affect their activity profile has been identified. These new proteins were discovered while studying the
expression of a complementary DNA that encoded a putative GPCR, which did not lead to the
expression of a functional receptor. Specifically, a cDNA named calcitonin-receptor-like receptor
(CRLR), which showed 55% overall identity to the calcitonin-receptor gene, was proposed to encode
the receptor for the calcitonin-gene-related peptide (CGRP). However, various attempts to show that it
was indeed the CGRP receptor failed because it was impossible to demonstrate any type of functional
expression.
6. GPCR dimerization…
Potential roles of G-protein-coupled receptor (GPCR) dimerization during the GPCR life
cycle.(1) In some cases, dimerization has been shown to have a primary role in receptor
maturation and allows the correct transport of GPCRs from the endoplasmic reticulum (ER) to
the cell surface. (2) Once at the plasma membrane, dimers might become the target for
dynamic regulation by ligand binding. (3) It has been proposed that GPCR heterodimerization
leads to both positive (+) and negative (-) ligand binding cooperativity, as well as (4) potentiating
(+)/attenuating (-) signalling or changing G-protein selectivity. (5) Heterodimerization can
promote the co-internalization of two receptors after the stimulation of only one protomer.
Alternatively, the presence of a protomer that is resistant to agonist-promoted endocytosis,
within a heterodimer, can inhibit the internalization of the complex. G, G protein; L, ligand.
EMBO Rep. 2004; 5:30-4.
6. GPCR dimerization…
Summary of studies on
hetero-dimerization in
different GPCR classes and
the possible functional roles
of GPCR dimerization
6. GPCR dimerization…
Summary of studies on heterodimerization in different GPCR
classes and the possible
functional roles of GPCR
dimerization (cont’d)
Summary of factors effecting
GPCR signalling
The coupling of GPCRs with multiple G-proteins is selectively regulated at different levels. First,
the nature of the response is dependent on the agonist used, which may selectively favour the
coupling with a subset of G-proteins. In addition, when multiple couplings occur, several studies
have demonstrated that the agonist elicited the responses with different potencies. Therefore,
the involvement of multiple G-proteins is influenced by the concentration of agonist. Secondly,
alterations in the expression or in the structure of the receptor have been shown to affect the
coupling profile with multiple G-proteins. Thus, distinct coupling properties have been observed
for related splice variants of a same receptor. Post-translational modifications, such as
palmitoylation and phosphorylation, are also involved in the dynamic regulation of the G-protein
coupling specificity. Many recent studies have shed light on the critical role played by GPCRinteracting proteins in determining the efficiency of coupling with distinct G-proteins. Finally, the
availability of distinct G-proteins and the selective modulation of their expression, localisation,
and activity contribute to determining the specificity of the intracellular signalling triggered after
the receptor activation.
Pharmacol Ther. 2003 99:25-44
7. Alternative functions of GPCRs
Figure 1. HIV-1 fusion according to
current models Env is composed of a
surface subunit gp120 and
transmembrane subunit gp41, which
are non-covalently associated and
then assembled as a trimer. The CD4
binding region of gp120 is exposed,
but variable regions of gp120 screen
important conserved structures
involved in chemokine receptor
interactions. Each gp41 molecule
contains two alpha-helices that form a
hairpin configuration, and the Nterminal hydrophobic fusion peptide is
buried within the complex. gp120
binds to CD4 (A) and undergoes
conformational changes that create or
unmask the co-receptor binding site
(B) and enable binding to the
chemokine receptor (C). Structural
changes are then induced in gp41 that
extend the helical domains to form a
‘pre-hairpin intermediate’ (D). The
hydrophobic fusion peptide inserts into
the target cell membrane, causing
gp41 to span between the virus and
cell membranes. The gp41 helices
then fold into a six-helix bundle,
bringing together the N-terminal and
C-terminal domains and thus the viral
and cellular membranes (E). Contact
between the membranes allows mixing
of the outer leaflets followed by the
development of a fusion pore (G).
gp120 is omitted from panels F and G
for the sake of clarity. Modified after
Starr-Spires and Collman [6].
From: Shaheen: Curr Opin Infect Dis,
Volume 17(1).February 2004.7-16
The Nobel Prize in Physiology or Medicine 2004
B1. Odorant receptors
Richard Axel
Linda B. Buck
1/2 of the prize
1/2 of the prize
USA
USA
Columbia University
New York, NY, USA;
Howard Hughes
Medical Institute
Fred Hutchinson
Cancer Research
Center
Seattle, WA, USA;
Howard Hughes
Medical Institute
b. 1946
b. 1947
"for their discoveries of odorant receptors and the organization of the olfactory system"
B1. Odorant receptors…
The sense of smell: genomics of vertebrate odorant receptors.
Olfactory receptor (OR) proteins interact with odorant molecules in the nose,
initiating a neuronal response that triggers the perception of a smell. The OR family
is one of the largest known mammalian gene families, with around 900 genes in
human and 1500 in mouse. After discounting pseudogenes, the functional
repertoire in mouse is more than three times larger than that of human. OR genes
encode G-protein-coupled receptors containing seven transmembrane domains.
ORs are arranged in clusters of up to 100 genes dispersed in 40-100 genomic
locations. Each neuron in the olfactory epithelium expresses only one allele of one
OR gene. The mechanism of gene choice is still unknown, but must involve locus,
gene, and allele selection. The gene family has expanded mainly by tandem
duplications, many of which have occurred since the divergence of the rodent and
primate lineages. Interchromosomal segmental duplications including OR genes
have also occurred, but more commonly in the human than the mouse family. As a
result, many human OR genes have several possible mouse orthologs, and vice
versa. Sequence and copy number polymorphisms in OR genes have been
described, which may account for interindividual differences in odorant detection
thresholds.
Hum Mol Genet. 2002,11:1153
B1. Odorant receptors…
Figure 32.2. The Main Nasal Epithelium. This region of the nose, which lies at the
top of the nasal cavity, contains approximately 1 million sensory neurons. Nerve
impulses generated by odorant molecules binding to receptors on the cilia travel
from the sensory neurons to the olfactory bulb.
Biochemistry.
Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert
The olfactory system
The olfactory epithelium contains millions of olfactory neurons, which
send messages directly to the olfactory bulb of the brain.
The olfactory receptor cells are the only neurons in the nervous
system exposed directly to the external environment.
B1. Odorant receptors…
Species differences
The area of the olfactory epithelium (red) in
dogs is some forty times larger than in
humans. Mice – the species Axel and Buck
studied – have about one thousand
different odorant receptor types.
Humans have a smaller number than mice;
some of the genes have been lost during
evolution. There are several millions of
olfactory receptor cells in our olfactory
epithelium.
A large family of odorant
receptors
Richard Axel and Linda Buck
published their fundamental paper in
1991, in which they described the
genes coding for a large family of
odorant receptors.
The odorant receptors are located on
the olfactory receptor cells in the
nasal cavity. Each olfactory receptor
cell expresses only one type of
odorant receptor, and each receptor
can detect a limited number of
odorant substances.
The olfactory receptor
Each receptor consists of a protein chain
that traverses the cell membrane seven
times.
When an odorant substance attaches to an
olfactory receptor, the shape of the
receptor protein is altered, leading to a G
protein activation.
An electric signal is triggered in the
olfactory receptor neuron and sent to the
brain via nerve processes.
Small variations
All odorant receptors are related proteins
and differ only in some amino acid
residues (indicated in green, blue and red).
The subtle differences in the protein chains
explain why the receptors are triggered by
different odorant molecules.
B1. Odorant receptors…
(A) The combinatorial code of olfaction.
Neurons expressing a given receptor can
respond to more than one type of odorant (e.g.
green receptor). Each odorant can elicit
responses from several receptors, perhaps with
different response amplitudes (here, the red
receptor reacts strongly and the green receptor
less strongly). Thousands of neurons
expressing a given olfactory receptor are
spread throughout one zone of the olfactory
epithelium, but their axons converge on one or
two glomeruli in the olfactory bulb. (B) Sources
of phenotypic variation in olfaction. Individuals
with different genotypes may (1) be
homozygous for a given olfactory receptor, (2)
express sequence variants with slightly different
odorant-binding capabilities, (3) possess nonfunctional variants (hatched receptor) and/or (4)
have duplicate gene copies, perhaps changing
relative numbers of responsive neurons in the
olfactory epithelium.
Human Molecular Genetics, 2002, 11, 1153
B1. Odorant receptors…
Genomic organization of olfactory receptor genes. Top: OR genes have a single
main coding exon (black) and typically have several 5' untranslated exons. Alternate
splicing is seen in many genes. Middle: OR genes are clustered in the genome in
groups of 1 to over 100 genes (green arrows) and pseudogenes (red arrows) in
both transcriptional orientations. Bottom: OR clusters are dispersed around the
genome in more than 40 (mouse) or over 100 (human) locations.
Models of odorant receptor (OR)
transcriptional regulation. (a) The short
promoter model. The sequences
immediately upstream of OR genes
contain transcription factor binding sites
sufficient to regulate receptor
transcription. Which OR gene is
expressed depends on the transcription
factor(s) expressed in the cell. (b) The
locus control region (LCR) model.
Proximal promoters contain transcription
factor binding sites necessary to drive
specific receptor transcription, but are
unavailable until factors binding to a distal
LCR makes the region transcriptionally
accessible. (c) The recombination model.
OR genes are translocated by
recombination or copied by gene
conversion into a single active locus for
expression. Transcription promoting
factors are depicted as ovals.
Trends Genet. 2002 Jan;18(1):29-34
B1. Odorant receptors…
Figure 1. Gene-translocation mechanisms that activate one member of the
multigene family. (a) DNA recombination. As seen in the mouse immunoglobulin
(Ig) κ light-chain genes, DNA deletion brings a promoter carried by each variable
(V) gene segment and the enhancer region between the joining (J) and constant
(C) gene segments into proximity, thus activating the translocated gene. (b)
Gene conversion. This is another gene-translocation mechanism that activates
one particular member of the multigene family. A copy of the gene to be
activated is transferred into the expression cassette located remotely from the
gene cluster. This activation mechanism can be found in the yeast mating-type
choice and antigenic variation in African trypanosomes. Abbreviations: P,
promoter; E, enhancer.
Trends Genet. 2004 Dec;20(12):648-53
Figure 32.5. The Olfactory Signal-Transduction Cascade. The binding of
odorant to the olfactory receptor activates a signaling pathway similar to those
initiated in response to the binding of some hormones to their receptors The
final result is the opening of cAMP-gated ion channels and the initiation of an
action potential.
Sensory transduction. Within the compact cilia of the OSNs a cascade of enzymatic
activity transduces the binding of an odorant molecule to a receptor into an electrical
signal that can be transmitted to the brain. This is a classic cyclic nucleotide
transduction pathway in which all of the proteins involved have been identified,
cloned, expressed and characterized. Additionally, many of them have been
genetically deleted from strains of mice, making this one of the most investigated
and best understood second-messenger pathways in the brain. AC, adenylyl
cyclase; CNG channel, cyclic nucleotide-gated channel; PDE, phosphodiesterase;
PKA, protein kinase A; ORK, olfactory receptor kinase; RGS, regulator of G proteins
(but here acts on the AC); CaBP, calmodulin-binding protein. Green arrows indicate
stimulatory pathways; red indicates inhibitory (feedback).
B1. Odorant receptors…
Combinatorial receptor codes
The odorant receptor family is used in a
combinatorial manner to detect odorants
and encode their unique identities.
Different odorants are detected by different
combinations of receptors and thus have
different receptor codes. These codes are
translated by the brain into diverse odour
perceptions.
The immense number of potential receptor
combinations is the basis for our ability to
distinguish and form memories of more
than 10,000 different odorants.
B1. Odorant receptors…
Smell
Molecule Name
Chemical
Formula
Fruity
ethyl octanoate
C10H20O2
Minty
beta-cyclocitral
C10H13O
Minty
p-anisaldehyde
C8H8O2
Nutty,Medicinal
2,6-dimethyl
pyrazine
C6H8N2
Nutty,Medicinal
4-heptanolide
C7H12O2
Nutty,Medicinal
p-cresol
C7H8O
Shape
B1. Odorant receptors…
Testes and the nose both express members of the odorant receptor super-family of
G protein-coupled receptors.The testes odorant receptor hOR 17-4, previously
shown to interact with the floral odor bourgeonal, is now shown to be expressed in
the nose as well. This suggests that hOR 17-4 has evolved a dual role in
chemoreception: perhaps guiding sperm to the egg and providing a conscious
perception of odors through the nose. A gradient of bourgeonal is shown to attract
sperm (left) and to be smelled by the nose (right).
Curr Biol. 2004 Nov 9;14(21):R918-20
B2-GPCRs as drug targets: orphan and known GPCRs
Schematic representation of the number and classification of liganded and
orphan GPCRs.
Annu Rev Pharmacol Toxicol. 2004;44:43-66.
B2- GPCRs as drug targets: orphan and known GPCRs…
Box 1 | Methods for 'de-orphanizing' seven-transmembrane (7TM) receptors Three main approaches have been taken to identify the
endogenous ligands for orphan receptors. First, peptides or other putative ligands that are known to have bioactivity in distant species have been
found to exist in mammals, and subsequently matched as agonists for orphan receptors. A second related approach has been to identify potential
peptides, either biochemically from tissue extracts or by predictive means using the sequences of apparent neuropeptide precursors. These
peptides are then synthesized and tested for bioactivity at orphan and known receptors. A distinct third approach has been to use the orphan
receptors themselves in functional screens of fractionated tissues, and those that gave rise to an 'active peak' (that is, they include a component
that binds to the receptor) are then sequenced or subjected to mass spectroscopy to identify the active principle. This approach is shown above.
Nature Reviews Molecular Cell Biology 3; 639-650 (2002);
B2- GPCRs as drug targets: orphan and known GPCRs…
Strategy for the identification of ligands at orphan GPCRs. Orphan GPCRs are expressed in a recombinant expression
system, such as mammalian cells, yeast, or Xenopus melanophores. Following expression, it is usual to generate an
assay amenable to the screening of candidate ligands in 96 well or 384 well microtitre plate formats. Candidate ligands,
including small molecules, peptides, proteins, lipids, or tissue extracts, are screened in the assay. The identification of an
activating ligand is detected according to the activation of an intracellular signaling cascade (see text for details). An
activating ligand, often termed a hit molecule, will be identified according to its ability to cause a concentrationdependent increase in the activity of a signaling cascade. Once identified, the ligand may be further characterized
against other GPCRs to determine its activity and selecticity profile prior to being used in cell-based, tissue, and in some
cases whole-animal experiments in order to study the physiological role of the newly liganded receptor.
Annu Rev Pharmacol Toxicol. 2004;44:43-66.
B2- GPCRs as drug targets: orphan and known GPCRs…
Characterization of HM74 as a Gprotein coupled receptor that responds
to nicotinic acid. (A) Application of
300 µM (hashed columns) and 1 mM
(filled columns) nicotinic acid to
membranes from HEK293/T cells
expressing a variety of orphan G
protein coupled receptors was found to
stimulate [35S]-GTPγS binding in
membranes from cells expressing
HM74 (open columns, basal
conditions). (B) Nicotinic acid
stimulated a dose-dependent increase
in [35S]-GTPγS binding in cells
expressing HM74.
Annu Rev Pharmacol Toxicol. 2004;44:43-66.
B2- GPCRs as drug targets: orphan and known GPCRs…
Recent peptide/GPCR pairings
Trends Pharmacol Sci. 2003 Jan;24(1):30-5
B2- GPCRs as drug targets: orphan and known GPCRs…
Natural ligand
Sites of action
G protein coupling profile
Kyotorphin
Nocistatin
Prosaposin
Chromostatin
Pancreastatin
CNS
Brain,spinal cord
Brain
Adrenal gland
Heart, liver, adipose
Gi/o
Gi/o
Gi/o
Gi/o
Gq/11>Gi/o
Examples of naturally occurring ligands that are likely to mediate
their biological effects via GPCRs
Annu Rev Pharmacol Toxicol. 2004;44:43-66.
Examples of Pharmaceutical GPCR research
Annu Rev Pharmacol Toxicol. 2004;44:43-66.
The identification of ligands at orphan G-protein coupled receptors.
Wise A, Jupe SC, Rees S.
7TMR Systems Research Europe, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY,
United Kingdom
FEBS Lett. 2005 Jan 3;579(1):259-64.
Identification and characterisation of a novel splice variant of the human CB1 receptor.
Ryberg E, Vu HK, Larsson N, Groblewski T, Hjorth S, Elebring T, Sjogren S, Greasley PJ.
Department of Molecular Pharmacology, AstraZeneca R&D, Molndal, Sweden
J Mol Graph Model. 2004 Sep;23(1):15-21. Design of a gene family screening library targeting Gprotein coupled receptors.
Lamb ML, Bradley EK, Beaton G, Bondy SS, Castellino AJ, Gibbons PA, Suto MJ, Grootenhuis PD.
Deltagen Research Laboratories, 740 Bay Rd., Redwood City, CA 94063, USA
J Med Chem. 2004 Sep 9;47(19):4677-83.
Indolebutylamines as selective 5-HT(1A) agonists.
Heinrich T, Bottcher H, Bartoszyk GD, Greiner HE, Seyfried CA, Van Amsterdam C.
Preclinical Pharmaceutical Research, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany.
Drug Discov Today. 2005 Jan 1;10(1):69-73
Predicting ligands for orphan GPCRs.
Huang ES.
Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139 USA
Recruitment, localisation, and tissue exit of circulating leucocytes. Tissue localisation
of leucocytes involves two distinct and sequential processes, termed extravasation
and chemotaxis. During extravasation, blood leucocytes interact with adhesion
molecules on the luminal side of blood vessels and, upon chemokine receptor
triggering, become firmly attached and transmigrate through the epithelial barrier.
Subsequent chemotaxis guides the perivascular leucocytes to the cellular source(s)
of chemokines, which enables cellular colocalisation and subsequent execution of
leucocyte function. Eventually, leucocytes exit the tissue via afferent lymphatic
vessels to reach draining lymph nodes (LNs) and peripheral blood.
Ann Rheum Dis. 2004 Nov;63 Suppl 2:ii84-ii89
Chemokines. Chemokines are bioactive peptides that regulate leukocyte activation and migration. They are essential mediators of
inflammation and are crucial for the control of viral infections. With around 50 members, the chemokine superfamily can be divided — on
the basis of the arrangement of the first highly conserved N-terminal cysteine residue (or residues) — into four distinct classes: CXC ( ),
CC ( ), CX3C ( ), and C ( ) (where C is cysteine and X is any amino acid; see figure). All chemokines are structurally similar, having at
least three
-pleated sheets and a C-terminal -helix, with disulphide bonds connecting these cysteine residues. There is a comparably
large number of chemokine receptors (19 in humans), and each chemokine class has a preference for a subfamily of these G-proteincoupled receptors (GPCRs), which are often expressed in different subsets of leukocytes as well as in other cells — for example, in
endothelial cells and neurons. CXC-chemokines bind preferentially to CXC-chemokine receptors (CXCRs) and attract neutrophils, whereas
CC-chemokines generally bind to CC-chemokine receptors (CCRs) and tend to attract monocytes, lymphocytes, eosinophils and BASOPHILS.
The C-chemokine, lymphotactin (XCL1), binds to XCR1 and induces neutrophil and T- and B-cell migration. Finally, the CX3C-chemokine,
fractalkine (CX3CL), is capable of functioning as a membrane-anchored ligand, or as a shed ligand, and binds CX3CR1 on blood-derived
neutrophils, monocytes, natural killer cells and T lymphocytes.
Leukocyte expression and ligand specificity of
chemokine receptors at a glance. Receptors, selected
cognate ligands and predominant receptor repertoires in
different leukocyte populations are listed. The selected
ligands are identified with one old acronym and with the
new nomenclature, in which the first part of the name
identifies the family and L stands for ‘ligand’ followed by
a progressive number. Red identifies predominantly
‘inflammatory’ or ‘inducible’ chemokines, green
‘homeostatic’ agonists, yellow molecules belonging to
both realms. Chemokine acronyms shown are as follows: BCA, B cell
activating chemokine; BRAK, breast and kidney chemokine; CTACK, cutaneous
T-cell attracting chemokine; ELC, Epstein–Barr virus-induced receptor ligand
chemokine; ENA-78, epithelial cell-derived neutrophil-activating factor (78 amino
acids); GCP, granulocyte chemoattractant protein; GRO, growth-related
oncogene; HCC, hemofiltrate CC chemokine; IP, IFN-inducible protein; I-TAC,
IFN-inducible T-cell α chemoattractant; MCP, monocyte chemoattractant protein;
MDC, macrophage-derived chemokine; Mig, monokine induced by gamma
interferon; MIP, macrophage inflammatory protein; MPIF, myeloid progenitor
inhibitory factor; NAP, neutrophil-activating protein; PARC, pulmonary and
activation-regulated chemokine; RANTES, regulated upon activation normal T
cell-expressed and secreted; SCM, single C motif; SDF, stromal cell-derived
factor; SLC, secondary lymphoid tissue chemokine; TARC, thymus and activationrelated chemokine; TECK, thymus expressed chemokine. Abbreviations: PMN,
neutrophils; Eo, eosinophils; Ba, basophils; MC, mast cells; Mo, monocytes; Mø,
macrophages; iDCs, immature dendritic cells; mDCs, mature DCs; T naïve, naïve
T cells; T act, activated T cells; T skin, skin-homing T cells; T muc, mucosalhoming T cells; Treg, regulatory T cells
Model of Chemokine Regulation of BreastCancer Metastasis.
Metastasis is an orderly, multistep process
involving the movement of cancer cells from
the primary tumor to specific organs under
the guidance of specific chemokines. First,
cancerous mammary epithelial cells
undergo clonal proliferation, invade local
tissue, induce angiogenesis, and express
CXC chemokine receptor 4 (CXCR4) on
their surface. Then, cancer cells detach
from the primary tumor, migrate across
lymphatic and vascular walls in the tumor,
and enter the systemic circulation. Cancer
cells are arrested in vascular beds in organs
that produce high levels of the CXCR4
ligand (CXCL12), which is expressed on the
surface of vascular endothelial cells.
Binding of CXCL12 to CXCR4 induces the
migration of cancer cells into normal tissue,
where the cells proliferate, induce
angiogenesis, and form metastatic tumors.
Breast-cancer cells do not usually
metastasize to organs that produce low
levels of CXCL12, such as the kidney.
N Engl J Med. 2001 Sep 13;345(11):833
Figure 1 | Viral pathogen hijacking of intracellular signalling networks is regulated by GPCRs. a |
On binding by chemokines (among many other agonists), cellular G-protein-coupled receptors (GPCRs) signal
through a complex system of intracellular pathways. b | Viruses (such as HIV) might use cellular GPCRs as coreceptors for viral entry; viral envelope glycoproteins (gps) might function as agonists or antagonists of
cellular GPCRs, modulating their downstream signalling pathways. c | Viruses might encode their own GPCRs,
which often constitutively signal to a network of intracellular cascades; cellular chemokines could function as
agonists or antagonists of viral GPCRs, thereby modulating these signalling networks. d | Virally encoded
chemokines (virokines) might also function as agonists or antagonists of cellular (or viral) GPCRs. e | Virally
encoded chemokine-binding proteins (CKBPs) bind to and sequester cellular chemokines, thereby preventing
the activation of cellular GPCRs by endogenous chemokines.
Nat Rev Mol Cell Biol. 2004;5:998-1012
Comparison of Kaposi's sarcoma-like lesion at the nose of a mouse and a
man. Kaposi's sarcoma-like lesion at the nose of a mouse transgenic
expressing ORF74 from HHV8 under control of the CD2 promoter (left side)
compared to a Kaposi's sarcoma lesion at a human nose
Oncogene. 2001; 20:1582-93.
Figure 2 | Signalling downstream of HIV gp120–CCR5
interactions. Stimulation of CC-chemokine receptor-5 (CCR5) by R5 glycoprotein
(gp)120 in macrophages activates proliferative signalling pathways such as the
mitogenic signalling cascade from Raf to MEK (mitogen-activated protein kinase
(MAPK) and extracellular signal-regulated kinase (ERK) kinase) to ERK. This thereby
promotes the proliferation of virally infected cells. Concurrently, stimulation of
stress-activated kinases (such as p38 and Jun N-terminal kinase (JNK), which are
downstream of MAPK kinase 3/6 (MKK3/6) and MEK kinase-1 (MEKK1), respectively;
see Box 2) might enhance the transcriptional activity of key transcription factors
(such as nuclear factor (NF)- B and activator protein-1 (AP-1)), and lead to the
secretion of cellular cytokines that could promote the recruitment of target cells or
further enhance infected-cell proliferation through paracrine mechanisms.
Collectively, these pathways could facilitate HIV replication and promote viral
transmission.
Figure 3 | Interaction of CXCR4 with HIV gp120 promotes lymphocyte
migration and apoptosis. Binding of CXC-chemokine receptor-4 (CXCR4) by X4
glycoprotein (gp)120 in T cells leads to the stimulation of focal adhesion kinase
(FAK) or protein tyrosine kinase-2 (PYK2), and SRC (left-hand pathway). When
these tyrosine kinases are stimulated, adaptor molecules such as CRK-associated
substrate (CAS), paxillin and CRK promote the recruitment/activation of the
engulfment and cell motility protein (ELMO)–Dock180 complex, which is a Rac
guanine nucleotide-exchange factor. ELMO–Dock180 activation triggers the
exchange of GDP for GTP by Rac. GTP-bound (active) Rac stimulates actin
polymerization and cell adhesion and migration through p21-activated kinase-1
(PAK1). Activation of FAK/PYK2 by gp120 binding to CXCR4 might also stimulate
signalling through phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent
protein kinase (PDK) and AKT/protein kinase B (AKT/PKB; right-hand pathway),
thereby promoting host-cell survival. In certain cases, however, CXCR4 activation
might lead to T-cell apoptosis — as depicted in Fig. 4 for neuronal cells. Collectively,
these pathways could help to ensure successful viral infectivity and propagation, and
promote disease progression. Similar pathways are also activated by R5 gp120
stimulation of CCR5 (not shown).
Figure 5 | Role of KSHV-GPCR signalling pathways in Kaposi's sarcomagenesis. a | The expression of Kaposi's-sarcoma-associated
herpesvirus (KSHV) G-protein-coupled receptor (GPCR) in KSHV-infected endothelial cells might be essential for triggering Kaposi's
sarcomagenesis. KSHV-GPCR-expressing cells release angiogenic growth factors that recruit (and transform) adjacent endothelial cells through
paracrine mechanisms. These endothelial cells might be latently infected with KSHV, which would further promote cell proliferation and survival.
b | Signalling pathways that are upregulated by KSHV GPCR in endothelial cells might induce endothelial-cell transformation through direct and
indirect (paracrine) mechanisms. KSHV GPCR, by releasing
subunits (Box 2), promotes signalling through phosphatidylinositol 3-kinase
(PI3K), phosphoinositide-dependent kinase (PDK) and AKT/protein kinase B (AKT/PKB). Concurrently, KSHV-GPCR-mediated activation of
mitogen-activated-protein-kinase cascades (such as Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38; Box 2)
through the small GTPases Ras and Rac1 stimulates the activity of key cellular transcription factors (such as activating protein-1 (AP-1),
nuclear factor (NF)- B, and hypoxia-inducible factor-1 (HIF1)). These transcription factors upregulate the transcription and secretion of proangiogenic growth factors (such as interleukin-8 (IL-8)/CXC-chemokine ligand-8 (CXCL8), Gro /CXCL1, and vascular endothelial growth factor
(VEGF)). Secreted growth factors might then bind to and activate endogenous cellular receptors, like kinase insert deaminase receptor (KDR),
which leads to the indirect (paracrine) activation of AKT/PKB. Several of these secreted cytokines could also function as agonists for KSHV
GPCR, enhancing its signalling through an autocrine mechanism.