Download Homogeneous GTP Binding Assay For G

Homogeneous GTP Binding Assay For G-protein Coupled
Receptors Based on TR-FRET
Heini Frang, Pertti Hurskainen, Veli-Matti Mukkala and Ilkka Hemmilä
PerkinElmer Life and Analytical Sciences, Wallac Oy, P.O.Box 10, FIN-20101 Turku, Finland
4
Assay protocol (final volume 40 µl/well):
- pipet all assay components into the white
384-microtitration plate:
* reaction buffer with muscarinic M2 receptor
contained 50 mM Hepes (pH 7.4), 10 µM GDP,
5 mM MgCl2, 10 mM NaCl and 0.05% TSA-BSA.
* M2 cell membrane concentration 10 µg/well
* KaroBio’s bio-peptide (10-40 nM)
* SA-Eu (W1024 or W8044 from PerkinElmer)
(0.5-5 nM)
* Alexa647-GTP (10-50 nM)
* ± different concentrations of suitable agonist
* ± 5 µM GTPγS to detect non-specific binding
* incubation at RT for ~30 min
* measured in VICTOR2TMV multilabel counter
using factory-set LANCE protocol
RESULTS
5 nM
3.5
For the TR-FRET GTP binding assay Alexa647
(Molecular Probes) was coupled to the γphosphate of GTP. Muscarinic M2 receptor was
over expressed with Gαi proteins in Sf9 cells and
the cell membrane fraction was used in the
assay. Biotinylated BioKey® peptide probe
recognizing the Gαi subunit in a GTP bound
conformation was kindly provided by KaroBio.
S/B ratio
G-protein coupled receptors (GPCRs) represent
one of the most important drug discovery
targets since the compounds targeted against
GPCRs form the largest drug class currently on
the market. General trend within HTS screening
arena favours functional assays when searching
new lead molecules for GPCR candidate drugs.
One considerable functional assay to monitor
GPCR activation is the cell membrane based
GTP binding assay. Agonist binding to GPCRs
stimulates the guanine nucleotide exchange,
GDP bound to Gα subunit of heterotrimeric Gproteins, dissociates and is replaced by GTP.
Karo Bio’s AlphaKey™ technology is based on
BioKey® peptide probes that e.g. specifically
bind to the Gαi subunit in a conformationdependent manner. In our TR-FRET model
system we have used biotinylated BioKey®
peptide recognizing only the GTP bound form of
the Gα subunit. Biotinylated peptide enables
binding of SA-Eu into close proximity of the
acceptor labelled GTP bound also to the Gα
subunit. TR-FRET GTP binding assay is a
homogeneous all-in-one assay highly
applicable to HTS screening for agonists of
GPCRs.
[bio-peptide]
4.0
5
10 nM
3.0
Figure legend: the effect of different bio-peptide :
SA ratios to the S/B ratio.
This optimization step was performed in the M2
receptor reaction buffer with 10 nM Alexa647GTP and 10 µg/wel cell membranes. Stimulation
was done with 100 µM bethanecol for 30 min at
RT.
20 nM
40 nM
2.5
2.0
1.5
1.0
0.8
1.6
3.2
[SA-Eu (W1024)] (nM)
6
Figure legend: dose-response curves with SA labelled with different Eu chelates (A) with SA-W1024 and B)
with SA-W8044). Assay conditions were: M2 receptor reaction buffer with 10 µg/wel cell membranes, 10 nM
Alexa647-GTP, 40 nM bio-peptide and 1 nM SA-Eu (W1024). B) Assay conditions like above except 50 nM
Alexa647-GTP and 0.8 nM SA-Eu (W8044) was used. Incubation times and EC50 values showed in the figure.
10 min; 16.3 µM
A
300
250
2
B
200
40 min; 6.7 µM
50 min; 8.2 µM
100
60 min; 4.7 µM
50
log [bethanecol] (M)
α
665 nm
Eu
βγ
BIO
SA
Schematic presentation of the assay
340 nm
7
60 min; 8.6 µM
50
0
-3
50 min; 10.3 µM
100
-50
-5
40 min; 12.4 µM
150
0
-7
30 min; 14.9 µM
200
-50
-9
20 min; 16.8 µM
250
30 min; 7.4 µM
150
10 min; 38.2 µM
300
20 min; 9.5 µM
% over basal
METHODS
3
INTRODUCTION
% over basal
1
-9
-7
-5
-3
log [bethanecol] (M)
CONCLUSIONS
• this new GTP binding assay based on TR-FRET and KaroBio’s conformation specific
peptide shows agonist concentration dependent signal increase which is replaceable
with GTPγS.
• we are still in the middle of optimization process and future goal is to be able to use
membranes from mammalian cell lines in the assay.
• assay is easily applicable to automation and therefore suitable for HTS screening.
PerkinElmer Life and Analytical Sciences, 710 Bridgeport Avenue, Shelton, CT USA (800) 762-4000 (+1) 203 925-4602 www.perkinelmer.com