Time-Resolved Fluorescence

Based GTP Binding Assay for

G-Protein Coupled Receptors

Gregory Warner, Rita Syystö, Heini Frang,

Christel Gripenberg-Lerche,
Jurgen Vanhauwe, Tarja Ahola,
and Satu Kovanen
 Abstract

G-protein coupled receptors (GPCRs) participate in a wide range of cell

signaling pathways and are believed to play a role in a variety of disease
states. Current screening efforts include not only receptor ligand binding
assays, but in addition functional assays including: Ca2+ flux, cAMP levels,
and GTP binding. Until now, GTP binding assays have revolved around the
use of the radioactive, non-hydrolyzable GTP analogue, [35S]GTPγS.
Time-resolved fluorometry (TRF) is a well-established technology that
exploits the unique fluorescence properties of lanthanide chelates to provide a
powerful alternative to radioisotopic assays in many HTS applications.
Using PerkinElmer Life Sciences’ labeling chemistry, we have developed a
non-radioactive, non-hydrolyzable, europium labeled GTP analogue, GTP-Eu,
for use in GPCR screening assays. The GTP-Eu binding assay yielded similar
if not better Z’ values and comparable EC50 values to the traditional
[35S]GTPγS filtration assay when tested using several GPCRs. Agonist-
induced stimulation of GTP-Eu binding above basal levels was at least
equivalent to and up to more than two times greater than that achieved by
the [35S]GTPγS assay. Using the PlateTrak™ robotic liquid handling system,
we have shown the ability to automate this assay without sacrificing
sensitivity or performance and estimate a minimum daily throughput of 200
plates. Thus, the end result is a highly sensitive, non-radioactive functional
assay to monitor GPCR activity through GTP binding that can be automated
and used in an HTS environment.

Introduction

Time-resolved fluorometry (TRF) is a well-established technology that

exploits the unique fluorescence properties of lanthanide chelates to provide
a powerful alternative to radioisotopic assays in many HTS applications.
TRF assays exhibit low background and high signal-to-noise ratios, two
attributes critical for robust HTS assays. Long fluorescence decay after
excitation allows time-delayed signal detection (microseconds) to virtually
eliminate all natural fluorescent background caused by cells and cell debris,
screening compounds, plates, and other reagents (half-life of nanoseconds).
A large Stokes shift (e.g., excitation and emission wavelengths for the
Eu-chelate are ~340 nm and ~615 nm, respectively) minimizes cross-talk,
resulting in a high signal-to-noise ratio. Therefore, because of their excellent
temporal and spectral resolution, lanthanide chelate labels can provide high
sensitivity assays.
New screening assays are continually being developed to measure G-protein
coupled receptors (GPCRs) activity. GPCRs participate in a wide range of
cell signaling pathways and are believed to play a role in a variety of
disease states. This in turn makes them attractive target for new therapeutics.
Currently around 30% of clinically prescribed drugs act on GPCR family
members. The application of TRF to GPCR assays to this point has mainly
been for receptor binding assays. Here we describe a new assay that allows
the user to examine GPCR activation by monitoring GTP binding through
GTP-Eu and take advantage of all the benefits of TRF technology.

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 Assay Principle

Materials:
• GTP-Eu • 10X GTP Wash Solution • AcroWell filter plates
• 5 M NaCl • 250 mM HEPES (pH 7.4) (Pall Gelman, Cat. #5020)
• 5 M MgCl2 • 2 mM GDP • Motilin (Bachem, Cat. #H-4385)
• 250 µM GTPγS • 50 mg/ml saponin • Neurotensin (Bachem, Cat. #H-4435)
• Oxotremorine M (Tocris, Cat. #1067)
• FMLF peptide (Bachem, Cat. #H-3030)
• Human Motilin Receptor membranes (PerkinElmer, Cat # RB-HMOTM)
• Human Neurotensin Receptor membranes (PerkinElmer, Cat # RB-HNT1M)
• Human Muscarinic M1 Receptor membranes (PerkinElmer, Cat # RB-HM1M)
• Human FPR1 Receptor membranes (BioSignal, Cat # 610527)
• [35S]GTPγS (1250 Ci/mmol) (PerkinElmer, Cat. # NEG030H)
Method:
• Assay Buffer: 50 mM HEPES, pH 7.4; 1-10 mM MgCl2; 0-200 mM NaCl; 0.1-10 µM
GDP; 0-1000 µg/ml saponin (components will be unique for each GPCR)
• Add 40 µl of assay buffer to all wells to be tested
• Add 35 µl of 50 mM HEPES, pH 7.4 to all wells to be tested
• Add 5 µl of agonist dilutions or 50 mM HEPES, pH 7.4 (blank) to all wells
being tested
• Add 20 µl of receptor membranes diluted in 50 mM HEPES to give 10 µg
membrane protein per well
• Incubate 30-60 min depending on receptor
• Add 10 µl of 100 nM GTP-Eu to all wells
• Incubate 10-30 min depending on receptor
• Filter and wash 2X with 300 µl of ice-cold 1X GTP Wash buffer
• Measure bound Eu at 615 nm using a time-resolved fluorometer such as VICTOR2™ V

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 1
120

GTP-Eu Binding
(% over basal)
100
80
60
40
20
10
0 5
MgCl2
0.1 1
1 (mM)
3
10
GDP (µM)

Figure 1. Optimization of MgCl2 and GDP concentrations for motilin-

induced binding of GTP-Eu to human motilin receptor. Human motilin
receptor (RBHMOT) (10 µg) was incubated in 50 mM HEPES (pH 7.4) with
various concentrations of GDP and MgCl2 in the presence or absence of 1
µM motilin for 30 min. GTP-Eu (30 nM) was added for an additional 30
min prior to filtration, washing, and measurement at 615 nm on VICTOR2V.

2 100
GTP-Eu Binding
(% above Basal)

75

50

25

0
0 20 50 100 200
N aC l ( m M)
Figure 2. Optimization of NaCl concentration for motilin-induced binding
of GTP-Eu to human motilin receptor. Human motilin receptor (RBHMOT)
(10 µg) was incubated in 50 mM HEPES (pH 7.4) 10 µM GDP, and 10 µM
MgCl2 with varying concentrations of NaCl in the presence or absence of 1
µM motilin for 30 min. GTP-Eu (30 nM) was added for an additional 30
min prior to filtration, washing, and measurement at 615 nm on VICTOR2V.

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 3
1 500 1500
GTP-Eu Binding
(% above Basal)

GTP-Eu Binding
(% above Basal)
1 000 1000

500 500

0 0
0 3 10 30 100 300 1 000 0 250 500 750 1000
Saponin (µg/ml)
Saponin (µg/ml)

Figure 3. Optimization of saponin concentration for motilin-induced

binding of GTP-Eu to human motilin receptor. Human motilin receptor
(RBHMOT) (10 µg membrane protein) was incubated in 50 mM HEPES
(pH 7.4) 10 µM GDP, 10 mM MgCl2, and 20 mM NaCl with varying
concentrations of saponin in the presence or absence of 1 µM motilin for
30 min. GTP-Eu (30 nM) was added for an additional 30 min prior to
filtration, washing, and measurement at 615 nm on VICTOR2V.

4 750
GTP-Eu Binding
(% above Basal)

500

250

0
0 1 4 7 10 13
Membrane protein (µg/well)

Figure 4. Optimization of human motilin receptor protein concentration.

Various amounts of human motilin receptor (RBHMOT) (0-13 µg membrane
protein) was incubated in 50 mM HEPES (pH 7.4) 10 µM GDP, 10 mM MgCl2,
20 mM NaCl, and 1000 mg/ml saponin in the presence or absence of 1 µM
motilin for 30 min. GTP-Eu (30 nM) was added for an additional 30 min prior
to filtration, washing, and measurement at 615 nm on VICTOR2V.

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 5 500

400
A.
[ 35S]GTPγS
GTP-Eu
1000
B.
[ 35S]GTPγS

% above basal
GTP-Eu

% above basal
750
300
500
200

100 250

0
0
10-12 10-10 10-8 10-6 10-4 10-12 10-10 10-8 10-6 10-4
Neurotensin (M) Motilin (M)
C.
200
C. [ S]GTPγ S
35

200 GTP-Eu
basalbasal

150 [ 35S]GTPγ S
150
GTP-Eu
100
% above

100
% above

50

50
0

0 10-9 10-7 10-5 10-3

-9
Oxotremorine
-7
M-5 (M)
10 10 10 10-3
Oxotremorine M (M)
Figure 5. Dose curves of agonist-induced binding of GTP-Eu and [35S]GTPγS
to GPCRs. Membranes expressing: A) human neurotensin; B) human
motilin, and C) human muscarinic M1 receptors were incubated with receptor
specific agonists under previously optimized conditions (data not shown).
Receptors were incubated with either 10 nM GTP-Eu or 0.2 nM [35S]GTPγS
for 30 min prior to filtration, wash, and counting on VICTOR2V (GTP-Eu) or
MicroBeta Jet ([35S]GTPγS).

6
25000

20000
(counts)
GTP-Eu

15000

10000

5000

0
Z’ EC50 (nM)
10-11 10-10 10-9 10-8 10-7 10-6 0.70 ± 0.11 19.2 ± 7.3
FMLF peptide (M)

Figure 6. Automation of GTP-Eu assay using the PlateTrak liquid handling

system. Six replicate plates of the GTP-Eu assay were performed on the
PlateTrak system using human FPR1 membranes (10 µg/well) and various
concentrations of FMLF peptide as the model assay. Membrane receptors
were incubated under the following buffer conditions: 50 mM Hepes, pH
7.4; 100 mM NaCl; 1 mM MgCl2; 10 µM GDP with 30 nM GTP-Eu for 30 min
prior to filtration and washing using the PlateTrak system. Subsequent
counting at 615 nm was performed on VICTOR2V.

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 7
RBHMOT RBHNT1 RBHM1
Receptor Motilin Neurotensin Muscarinic M1
Cell line HEK-293 HEK-293 CHO-K1
Agonist Motilin Neurotensin Oxotremorine M
50 mM HEPES 50 mM HEPES 50 mM HEPES
20 mM NaCl 10 mM NaCl 20 mM NaCl
Optimized buffer 10 mM MgCl2 10 mM MgCl2 10 mM MgCl2
10 µM GDP 1 µM GDP 0.1 µM GDP
100 µg/mL saponin 100 µg/mL saponin
EC50(1) (GTP-Eu) 30-40 nM 2.6–6.4 nM 2.3-6 µM
8 nM 3.8 nM; 0.7 µM;
EC50(1) ([35S]GTPγS) 2.3 ± 0.9 nM (3) 7.9 µM (4)
% above basal at EC50 value 260 200 120
(GTP- Eu)
% above basal at EC50 value 300 60 25
([35S]GTPγS)
Z’(2) (GTP-Eu) 0.63 0.67 0.32
Z’(2) ([35S]GTPγS) 0.44 0.62 0.29
(1) EC50 values have been measured at PerkinElmer Life Sciences’ Turku and Boston sites
(2) Z’ = 1-[(6*S.D.avg)/ (Avg.high-Avg.low)]
(3) Hermans et al. (1997) Br. J. Pharm. 121:1817-1823
(4) DeLapp et al. (1999) J. Pharm. Exp. Ther. 289:946-955

Conclusions

• Easy to optimize, non-radioactive GTP binding assay

• Performs as well or better than traditional [35S]GTPγS filtration assay in
terms of Z’ values obtained for dose curves of agonist-induced binding
• Agonist-induced stimulation of GTP-Eu binding above basal levels was
at least equivalent to and up to more than two times greater than that
achieved by the [35S]GTPγS assay
• Benefits of TRF assay format including reduced compound fluorescence
interference
• Can be automated as exemplified using PlateTrak liquid handling system
with an estimated daily throughput of 200 plates

PlateTrak and VICTOR are trademarks of PerkinElmer Life Sciences.

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