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Study of GPCR pharmacology using the DiscoveRx HitHunter™

cAMP HS assay on the FLUOstar OPTIMA
Julie M.-N. Rainard, Stewart E. Mireylees and Mark G. Darlison
School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK

Application Note 143

Rev. 09/2006

The assay directly measures the activity of G-protein coupled
receptors (GPCRs), coupled to either Gi or Gs proteins
The assay is sensitive and can detect low levels of cAMP (ideal
for cell-lines expressing endogenous receptors)
The assay can be used with the FLUOstar OPTIMA, a versatile reader
that can measure luminescence, absorbance and fluorescence
The reader accepts different assay formats from 6- to 1536-well plates
(suitable for academic environments and high-throughput screening; HTS)

Materials and Methods

G-protein coupled receptors (GPCRs) are cell surface receptors, which
represent the most predominant drug targets. Following stimulation
of these receptors, intracellular signalling pathways are activated and
this leads to a decrease (coupling to a Gi protein) or increase (via Gs or
Gq proteins) in the production of intracellular second messengers. The
common way of determining the activity of compounds is by measuring
the cellular formation of second messengers such as cAMP and calcium.
DiscoveRx assays offer a non-radioactive alternative for the detection
of a decrease or increase in second messenger production in cells.
They can be used with both cell-lines that express native receptors
and cells transfected with a GPCR of interest, and can be employed in
conjunction with high-throughput screening (HTS).
The HitHunter™ cAMP High Sensitivity (HS) assay is able to measure
low cAMP levels and is, therefore, particularly suitable for cell-lines
that endogenously express receptors at a level much lower than in
transfected cells overexpressing a cloned GPCR.
HitHunter™ cAMP assays are in vitro-based competitive
immunoassays that rely on enzyme fragment complementation
technology (EFC, Fig. 1).



Labeled ED

Inactive EFC

[Competing cAMP]


The purpose of this Application Note is to explain how to set up the
FLUOstar OPTIMA, for use with the HitHunter™ cAMP HS assay, to
detect changes in cAMP levels following agonist stimulation of cells
in the absence and presence of receptor antagonists. In this study, we
used the rat PC12 phaeochromocytoma cell-line which endogenously
expresses adenosine A2A and A2B receptors (Florio et al, 1999).
Both of these couple to Gs proteins, which promotes an increase in
intracellular cAMP levels upon receptor stimulation. Here, we have
utilised compounds selective for the A2A receptor.


Active EFC


All materials were purchased from the manufacturers stated.
HitHunter™ cAMP HS kit reagents (DiscoveRx):
Lysis buffer and antibody mixture
ED reagent
EA reagent and CL substrate mixture
cAMP standard (0.25mM)
Other reagents and materials:
Rat PC12 phaeochromocytoma cell-line (ATCC)
Dulbecco’s Phosphate Buffered Saline (PBS ; Cambrex)
Microplate, low volume, white with clear bottom, tissue culture
treated, sterile, 96-well (Corning)
Agonist (CGS21680; Tocris)
Antagonist (ZM241385; Tocris)
IBMX (Sigma; optional)
A full description of the use of the HitHunter™ cAMP assay is included
with the kit.
Low volume 96-well microplates were used. These allow the user to
reduce the cost of each experiment by half, by using the volumes of
reagents for a 384-well format.
To produce the standard curve, the cAMP standard provided in the
kit was diluted 1 in 25 to prepare the highest working concentration,
which was then used to prepare 1 in 3 serial dilutions in PBS giving
a range of concentrations from 2.7 × 10-6M to 4.6 × 10-11M cAMP in a
final assay volume of 55 µL. PBS alone was used as the control. PBS
was also used, with PC12 cells, to measure the cAMP produced by
constitutive receptor activity (basal activity).
Assay protocol (low volume 96-well plate):


Fig. 1: The Hit Hunter™ cAMP assay principle

Free cAMP molecules from cell lysates compete for antibody binding
with a labelled enzyme donor (ED)-cAMP conjugate, which contains
a small peptide fragment of ß-galactosidase. In the absence of free
cAMP, the ED-cAMP conjugates are captured by the cAMP-specific
antibody and are unavailable for complementation with the enzyme
acceptor (EA), resulting in a low signal. In the presence of free cAMP,
antibody sites are occupied, allowing the ED-cAMP conjugate to
complement with EA, forming an active ß-galactosidase enzyme;
substrate hydrolysis by this enzyme produces a chemiluminescent
signal. The signal generated is in direct proportion to the amount of
free cAMP bound by the antibody (Eglen, 2002). Any luminescence
reader used with the HitHunter™ cAMP HS assay has to be sensitive
enough to detect small changes in cAMP levels. The BMG LABTECH
FLUOstar OPTIMA microplate reader has this required sensitivity.

PC12 cells were seeded 48 hours prior to the experiment at a density
of 20,000 cells per well.
1. Add cAMP standard dilutions (15 µL) to empty wells of the microplate
2. Remove media from the cells and resuspend them in PBS
containing 500µM IBMX (10 µL)
3. Add antagonist to the cells (5 µL)
4. Incubate at 37°C for 15 min
5. Add agonist to the cells (5 µL)
6. Incubate at 37°C for 30 min
7. Add 10 µL of Lysis buffer and antibody mixture to each well
8. Incubate at room temperature for 60 min
9. Add 10 µL of ED reagent to each well
10. Incubate at room temperature for 60 min
11. Add 20 µL of EA reagent and CL substrate mixture to each well
12. Incubate at room temperature for at least 60 min
Chemiluminescence is then read on the FLUOstar OPTIMA 4 hours
after addition of the last reagent.

VI. and data from standard and agonist curves were obtained from the FLUOstar OPTIMA (BMG LABTECH) in luminescence mode. (2002). Fig. which endogenously expresses info@bmglabtech. F. A rightward shift of the agonist dose-response curve. BMG LABTECH SARL BMG LABTECH JAPAN Ltd. Dose-response curves for the selective A2A receptor agonist CGS21680 either alone or in the presence of the A2A receptor selective antagonist ZM241385 were generated (Fig.bmglabtech. 2: Read mode: Positioning delay: No. ASSAY and Drug Development Technologies 1. Gaion. The FLUOstar OPTIMA. 0 control -11 -10 -9 -8 log [cAMP. which can also be used for absorbance and fluorescence detection. M] -7 -6 -5 were measured in triplicate) Fig. Labmate (2006) Vol. 2: Screenshot of the settings window from the FLUOstar OPTIMA multimode reader for the HitHunter™ cAMP HS assay The HitHunter™ cAMP HS assay is particularly suitable for detecting small changes in cAMP levels such as those seen in. M. 3: cAMP standard curve for the HitHunter™ cAMP HS assay (standards 3000 pEC50 = 8(9. pEC50 values were calculated. Both agonist and antagonist data can be generated.g. Conclusion Fig. RLU 2000 The views expressed herein are those of the authors and do not necessarily represent those of Nottingham Trent University. 1000 First published in Intl. This shows that ZM241385 non-competitively antagonised (by 4. . Germany: BMG LABTECH GmbH Tel: +49 781 96968-0 Australia: France: Japan: UK: USA: Internet: BMG LABTECH Pty. It offers user-friendly software for both protocol set up and data analysis. Vertua. 3 illustrates the cAMP standard curve obtained using luminescence detection in a 96-well format. for example. 97-104. was observed in the presence of 10-7M ZM241385. using GraphPad Prism .7nM) S/B = 11 References .7±0. 817-824. in conjunction with the FLUOstar OPTIMA microplate reader. BMG LABTECH Inc. Ltd. Data was evaluated using Microsoft Excel™ in conjunction with the FLUOstar OPTIMA Excel™ evaluation package and the software package GraphPad Prism. each performed in triplicate. and shown in Fig. Tel: +61 3 59734744 Tel: +33 1 48 86 20 20 Tel: +81 48 647 7217 Tel: +44 1296 336650 Tel: +1 919 806 1735 www.32±0. Eglen. Results and Discussion The HitHunter™ cAMP HS assay from DiscoveRx was used in 96-well format..M] Fig. (1999). Amplification of cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A2A purinoceptors. BMG LABTECH Ltd. 5-fold) the agonist-induced increase in intracellular cAMP levels. the rat PC12 phaeochromocytoma cell-line. The curve shows a dose-dependent increase with a good signal-to-background noise (S/B) value of 11. It is very amenable for use in an academic environment (e. using the HitHunter™ cAMP HS assay. of kinetic windows: No. 4: Dose-response curves for CGS21680 in the presence or absence of ZM241385. Frausin. C. and a decrease in the maximal response. of multichromatics: Emission filter: Gain: Measurement interval time: pEC50 = 6.2µM) 1500 Plate 0.2 sec 1 1 lens 3000 1 sec RLU 2000 1000 500 0 OGS21680 +10-7M ZM241385 basal -10 -9 -8 -7 -6 -5 -4 log [CGS21680.06 (485nM) CGS21680 alone pEC50 = 5. Florio. R. 96-well format) but can also be employed in all formats up to 1536-well plates for HTS of library compounds. 4).FLUOstar OPTIMA settings: Basic parameters for luminescence plate mode detection are listed below. and chemiluminescence was read 4 hours after the addition of the last reagent. from three individual experiments. Journal of Pharmacology and Experimental Therapeutics 290. Enzyme fragment complementation: a flexible high throughput screening assay technology. was used here in luminescence mode.. Reagents were added according to the manufacturer’s protocol. XXXI. Is. R.. M.14 (2.