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Study of GPCR pharmacology using the DiscoveRx HitHuntercAMP HS assay on the FLUOstar OPTIMAJulie M.-N. Rainard, Stewart E. Mireylees and Mark G. DarlisonSchool 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 coupledreceptors (GPCRs), coupled to either Gi or Gs proteins

The assay is sensitive and can detect low levels of cAMP (idealfor cell-lines expressing endogenous receptors)

The assay can be used with the FLUOstar OPTIMA, a versatile readerthat 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)

Introduction

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 andthis leads to a decrease (coupling to a Giprotein) or increase (via Gsor

Gqproteins) 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 competitiveimmunoassays that rely on enzyme fragment complementation

technology (EFC, Fig. 1).

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 -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 -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.

Materials and Methods

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)

Dulbeccos 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 L. 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):

PC12 cells were seeded 48 hours prior to the experiment at a density

of 20,000 cells per well.

1. Add cAMP standarddilutions (15 L) to empty wells of the microplate2. Remove media from the cells and resuspend them in PBS

containing 500M IBMX (10 L)

3. Add antagonist to the cells (5 L)4. Incubate at 37C for 15 min

5. Add agonist to the cells (5 L)6. Incubate at 37C for 30 min

7. Add 10 L of Lysis buffer and antibody mixtureto each well8. Incubate at room temperature for 60 min

9. Add 10 L of ED reagentto each well10. Incubate at room temperature for 60 min

11. Add 20 L of EA reagent and CL substrate mixtureto each well12. 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.

The purpose of this Application Note is to explain how to set up theFLUOstar 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 Gsproteins, which promotes an increase in

intracellular cAMP levels upon receptor stimulation. Here, we have

utilised compounds selective for the A2Areceptor.

cAMPAntibody

Substrate

Hydrolyzed

Substrate Signal

Signal

[Competing cAMP]LabeledcAMP

CompetingcAMP

Inactive EFC

Enzyme

Active EFCEnzyme

ED

ED

EA

EA

+

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Germany: BMG LABTECH GmbH Tel: +49 781 96968-0

Australia: BMG LABTECH Pty. Ltd. Tel: +61 3 59734744France: BMG LABTECH SARL Tel: +33 1 48 86 20 20Japan: BMG LABTECH JAPAN Ltd. Tel: +81 48 647 7217UK: BMG LABTECH Ltd. Tel: +44 1296 336650USA: BMG LABTECH Inc. Tel: +1 919 806 1735

Internet: www.bmglabtech.com info@bmglabtech.com

Conclusion

The HitHunter cAMP HS assay from DiscoveRx was used in 96-well

format, and data from standard and agonist curves were obtained

from the FLUOstar OPTIMA (BMG LABTECH) in luminescence mode.

Reagents were added according to the manufacturers protocol, and

chemiluminescence was read 4 hours after the addition of the last

reagent.

Data was evaluated using Microsoft Excel in conjunction with theFLUOstar OPTIMA Excel evaluation package and the software pack-

age GraphPad Prism.Fig. 3: cAMP standard curve for the HitHunter cAMP HS assay (standards

were measured in triplicate)

Fig. 3 illustrates the cAMP standard curve obtained using luminescence

detection in a 96-well format. The curve shows a dose-dependent in-

crease with a good signal-to-background noise (S/B) value of 11.

Dose-response curves for the selective A2Areceptor agonist CGS21680

either alone or in the presence of the A2Areceptor selective antagonist

ZM241385 were generated (Fig. 4).

References

- Eglen, R. M. (2002). Enzyme fragment complementation: a flexible

high throughput screening assay technology. ASSAY and Drug De-

velopment Technologies 1, 97-104.

- Florio, C., Frausin, F., Vertua, R., Gaion, R. M. (1999). Amplification

of cyclic AMP response to forskolin in pheochromocytoma PC12

cells through adenosine A2A purinoceptors. Journal of Pharmacol-

ogy and Experimental Therapeutics 290, 817-824.

The views expressed herein are those of the authors and do not nec-

essarily represent those of Nottingham Trent University.

First published in Intl. Labmate (2006) Vol. XXXI, Is. VI.

FLUOstar OPTIMA settings:Basic parameters for luminescence plate mode detection are listed

below, and shown in Fig. 2:

Read mode: Plate

Positioning delay: 0.2 sec

No. of kinetic windows: 1

No. of multichromatics: 1

Emission filter: lens

Gain: 3000

Measurement interval time: 1 sec

Fig. 2: Screenshot of the settings window from the FLUOstar OPTIMA multi-mode reader for the HitHunter cAMP HS assay

Fig. 4: Dose-response curves for CGS21680 in the presence or absence ofZM241385. pEC50values were calculated, using GraphPad Prism soft-

ware, from three individual experiments, each performed in triplicate.

A rightward shift of the agonist dose-response curve, and a decrease

in the maximal response, was observed in the presence of 10 -7M

ZM241385. This shows that ZM241385 non-competitively antago-

nised (by 4- to 5-fold) the agonist-induced increase in intracellular

cAMP levels.

The HitHunter cAMP HS assay is particularly suitable for detecting

small changes in cAMP levels such as those seen in, for example,

the rat PC12 phaeochromocytoma cell-line, which endogenously

expresses GPCRs.

Both agonist and antagonist data can be generated, using the Hit-

Hunter cAMP HS assay, in conjunction with the FLUOstar OPTIMA

microplate reader.

The FLUOstar OPTIMA, which can also be used for absorbance and

fluorescence detection, was used here in luminescence mode. It of-

fers user-friendly software for both protocol set up and data analysis.

It is very amenable for use in an academic environment (e.g. 96-well

format) but can also be employed in all formats up to 1536-well

plates for HTS of library compounds.

Results and Discussion

RLU

log [cAMP, M]

3000

2000

1000

0

control -11 -10 -9 -8 -7 -6 -5

pEC50= 8(9.7nM)

S/B = 11

RLU

2000

1500

1000

500

0basal -10 -9 -8 -7 -6 -5 -4

log [CGS21680,M]

pEC50= 6.320.06 (485nM)

pEC50= 5.70.14 (2.2M)

CGS21680 alone

OGS21680+10-7M ZM241385