Signalling through GPCRs
Benjamin Smith , Marcin Jurdzinski & Graham Ladds
What happens if effector molecules are in
abundance?
Kinetic effects in
signal regulation
GPCR Signalling
Activity
Figure 5. Simulation of
GTPase cycle in the presence
and absence of RGS. Initial
conditions were set such that
effector molecules are always
available to be activated. The
curves begin to meet in the
plateau region.
Signalling under normal circumstances
At Low Ligand Concentration:
•Low levels of GPCR stimulation lead to a low rate of Gα
activation. The RGS can hydrolyse Gα –GTP as fast it is
produced so little signalling occurs.
(GPCR)
At High Ligand Concentration
•High levels of GPCR stimulation lead to a high rate of guanine
nucleotide exchange on Gα subunits. RGS cannot then
overcome the large number of Gα –GTPs so effector molecules
are activated and a response occurs.
Figure1. Types of signalling in a mammalian cell. There are many different types
of signalling system in mammalian cells. We study signalling through G Protein
Coupled Receptors (GPCRs), highlighted in red. They are a ubiquitous family of 7span transmembrane receptors.
-2
160.000
LacZ Activity
120.000
DeltaRgs1
100.000
Rgs1 (Wild
Type)
80.000
•60% of all prescription drugs (with a value of >$200 billion per year)
target GPCRs.
3
10
30
100
300
•Figure 3 shows a surprising result. RGS proteins can reduce
signalling at low levels of ligand stimulation but increase it at high
levels.
•This has variously been observed in GPCR signalling (e.g. G
protein gated K+ channels [Fujita et al. 2000, Ulens et al. 2000]
and pheromone response pathway in the yeast
Schizosaccharomyces pombe (Figure 3)) but no satisfactory
explanation exists.
Gα
•Experiments to test this are very difficult to design.
•By modelling the GTPase cycle as a dynamical system it should
be possible to investigate this phenomenon more thoroughly.
Figure 6. Simulation of
GTPase cycle in the presence
and absence of RGS. The rate
constants involved in the
15000
hydrolysis, autohydrolysis and
effector activation reactions
10000
were experimented with. The
deltaRGS
5000
autohydrolysis rate constant
RGS
(R4) is ~100 fold lower than
Log [Ligand] RGS catalysed hydrolysis
-1
1
2
(R6&7). Activation of effector is
fast. Binding of Gα-GTP to effector is slower when Gα-GTP is bound to RGS.
Now the graphs begin to display the behaviour we expect.
Activity
Figure 7. Simulation of
GTPase cycle in the presence
and absence of RGS. By
setting the rate constants such
that binding of Gα-GTP to
inactive effector (R8), happens
faster than subsequent
activation of effector (R9), GαGTP is sequestered if no RGS
is available to recycle it. The
curves display the behaviour
seen in experiment.
7000
6000
5000
4000
3000
deltaRGS
2000
RGS
1000
Log [Ligand]
-1
-0.5
0.5
1
1.5
2
•Through a process of questioning and simulation (Figures 4-7) a
detailed understanding of the GTPase cycle was reached.
•The simulation produces good qualitative agreement with
experimental data indicating that in Sz. pombe downstream
components have little effect on the extent of the signalling
response.
•The model now needs to be validated through further experiment.
Experimental Validation
The insight provided by working with the mathematical model
prompted the design of some biologically relevant experiments:
GTP
GDP
2
1000
•The effect is suspected to be due to the kinetics of the system,
with the components we have identified in the GTPase cycle being
sufficient to produce it.
GPCR
1.5
[Ligand]
•They are implicated in most diseases.
Premature births, cardiac disease, cancer, neurological
β γ
1
Is there a sequestering effect?
1
Figure 3. Graph of β-galactosidase activity (response to stimulation)
vs. concentration of ligand with which Schizosaccaromyces pombe
cells were stimulated. Sz. pombe express only one GPCR pathway,
with a GTPase cycle very similar to that shown in Figure 2. Sz.
pombe RGS is called Rgs1. The Magenta curve shows the dose
response of wild type cells. The Blue curve shows the dose response
of cells lacking RGS protein (delta Rgs1). The two curves very
clearly cross at high levels of stimulation (highlighted in red), with
the RGS apparently increasing signalling in this region. This result
seems counterintuitive, how can “switching off” Gα lead to
increased signalling?
disorders.
β γ
Gα
0.5
40.000
0
GPCR
-0.5
60.000
0.000
ACTIVATION
RGS
15000
20000
180.000
20.000
Ligand
deltaRGS
17500
How do hydrolysis rates of Gα-GTP compared
to rates of activation of effector affect
signalling?
200.000
•They interact with most intracellular effector systems, including ion
channels and transcription factors
The GTPase cycle
20000
Activity
140.000
How is GPCR signalling regulated?...
22500
Log [Ligand]
•Maximum levels of receptor stimulation lead to activation of all
available Gα. At this point signalling plateaus. Further increase
in ligand concentration fails to increase signalling. It is
sometimes hard to reach this plateau stage in experiments as
such a high response can be lethal to cells
•Human cells express a complex network of GPCR signalling
pathways. Some 400 non-sensory receptors exist (and >500
olfactory receptors)
25000
-1
At Very High Ligand Concentration
•Cells live and die by signalling. A large proportion of a cells energy
is devoted to processing information about its extracellular
environment and translating this into an adaptive response.
Signalling through GPCRs (highlighted in Figure 1) plays a vital role
in this process [Ladds et al., 2005].
27500
Overexpression of Gα
GTP autohydrolysis
•Effects of overexpression of Sz. pombe Gα on signalling can be
compared with predictions made by simulation.
GTP hydrolysis
Fast and Slow Cycling Mutants
RGS
DEACTIVATION
RESPONSE
Figure 2. Schematic of a basic GPCR signalling system. A heterotrimeric G
protein (Gαβγ) associates with a transmembrane GPCR to form a pre-activation
complex (top left). When ligand binds to GPCR a conformational change occurs
in the receptor allowing the Gα subunit to release GDP and bind GTP, thus
causing activation (top right). The activated G protein can then bring about a
response by interacting with downstream effector molecules (bottom right).
Hydrolysis of Gα -GTP returns the Gα subunits to their inactive state, thereby
terminating the signal. This hydrolysis reaction is catalysed by a class of proteins
called the Regulators of G protein Signalling (RGS).
•Figure 2 shows the basic unit of GPCR signalling, whereby
binding of a ligand to a receptor brings about a response in cells
through guanine nucleotide exchange on a Gα subunit [Ladds et
al., 2005] .
•In some systems the Gβγ subunit interacts with downstream
effector molecules to produce a response.
• The above reaction scheme describes all the reactions we
believe occur in the GTPase cycle. It was turned into a set of
ordinary differential equations.
•These were implemented in Mathematica™ and solved by
numerical integration.
• The resulting time domain data was used to output a simulation
of the dose response, resembling the biological system used in
experiment.
•In systems of equations such as this, the rate constants for each
of the reactions as well as the initial concentrations of the chemical
species determine the qualitative behaviour of the system. Initially
these were set with our best guess for relative values and the
simulation was run.
Activity
•Signalling is regulated by GTPase Activating Proteins known as
Regulators of G protein Signalling (RGS) [Dohlman and Thorner,
1997].
1.2
1
0.8
• In mammalian systems, where a number of different classes of G
protein, GPCR and RGS exist and may interact, the picture quickly
becomes complex.
0.6
deltaRGS
0.4
RGS
0.2
Log [Ligand]
Is the basic GTPase cycle fully understood?...
-1
-0.5
0.5
1
1.5
2
Figure 4. Simulation of
GTPase cycle in the presence
and absence of RGS.
Parameters and initial
conditions were set to values
that seemed sensible. The
resulting graphs do not
produce the crossing over
effect seen in experiment.
•Sequence comparison with other G proteins indicates that
specific single point mutations can produce fast and slow cycling
versions of Sz. pombe Gα subunit which can be cloned into the
yeast and assayed for effects on signalling. Results will be
compared with identical in silico experiments.
Future Work
•Move into looking at rat heart cells and human myometrium.
•Investigate the control, tolerance and exploitation of noise in
the Sz. pombe system.
Selected References
1.Didmon, M., K.Davis, P.Watson, G.Ladds, P.Broad, and J.Davey. 2002. Identifying regulators of
pheromone signalling in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 41:241-253.
2.Dohlman, H.G. and J.Thorner. 1997. RGS Proteins and Signaling by Heterotrimeric G Proteins. J. Biol.
Chem. 272:3871-3874.
3.Fujita, S., A.Inanobe, M.Chachin, Y.Aizawa, and Y.Kurachi. 2000. A regulator of G protein signalling
(RGS) protein confers agonist-dependent relaxation gating to a G protein-gated K+ channel. Journal of
Physiology-London 526:341-347.
4.Ladds, G., A.Goddard, and J.Davey. 2005. Functional analysis of heterologous GPCR signalling
pathways in yeast. Trends Biotechnol. 23:367-373.
5.Ulens, C., P.Daenens, and J.Tytgat. 2000. Changes in GIRK1/GIRK2 deactivation kinetics and basal
activity in the presence and absence of RGS4. Life Sci. 67:2305-2317.