Time-Resolved Fluorescence BasedGTP Binding Assay for Gs-Protein

Coupled Receptors

Patricia Kasila, Harry Harney, and Gregory Warner

PerkinElmer Life and Analytical Sciences, Boston MA, USA

2PerkinElmer Life and Analytical Sciences

Abstract

G-protein coupled receptors (GPCRs) participate in a wide range of cell signalingpathways and are believed to play an important role in a variety of disease states.Receptor ligand binding assays in the past have often been used in high throughputscreening (HTS) to develop therapeutics for GPCR targets. However, functional assayssuch as cAMP, Ca2+ flux and GTP binding are becoming significant technologies in theHTS laboratory to measure efficacy of compounds. Functional assays can detect theligand binding event as well as the functionality of the binding event. When doing GTPbinding assays, the response from a receptor ligand-binding event activates the GPCR,which in turn exchanges bound GDP for GTP. Recently a new technology has beenintroduced which allows GTP binding detection in a non-radioactive assay that is suitablefor HTS. The DELFIA GTP Binding Assay Kit from PerkinElmer Life and AnalyticalSciences has successfully been used to demonstrate GPCR functional assays for Gi andGq-protein coupled receptors. Gs-protein coupled receptors are more problematic dueto the slower hydrolysis rate of GTP compared to Gi-coupled receptors. Therefore, theturnover of inactive Gs to active Gs is much slower causing a decrease in the amountof GTP binding that occurs. PerkinElmer Life and Anayltical Sciences has optimizedconditions to successfully format a Time Resolved Fluorescence Gs -protein coupledreceptor functional assay yielding acceptable results.

1

PerkinElmer Life and Analytical Sciences

3

Introduction2

Materials

DELFIA GTP Binding Assay Kit PerkinElmer, Cat. #AD0167

10X GTP Wash Solution

5 M NaCl

250 mM HEPES (pH 7.4)

1 M MgCl2

GDP (lyophilized)

GTPgS (lyophilized)

Eu-GTP (lyophilized)

50 mg/ml saponin

AcroWell filter plate

Human Beta 2 Adrenergic Receptor PerkinElmer, Cat. #RBHBE2M

Epinephrine Sigma, Cat. #E4250

3

Add to Acro Wellfilter plate:

buffer componentsGPCR membranescompounds

Add GTP-Eu Filter & wash Measure at 615 nm

4PerkinElmer Life and Analytical Sciences

Methods

Add in the following order to AcroWell filter plate:

• 40 µl of β2 adrenergic receptor membranes

• 20 µl of 5X conc. agonist or buffer blank

• 40 µl of 10 nM Eu-GTP

• Incubate 60 min or indicated time

• Filter and Wash twice with 300 µl of 1X GTP wash buffer

• Read 615 nm emission on Victor2V, EnVision, ViewLux or other TRF enabled reader

4

Results5

GDP was titrated to final concentrations of 0.1, 1, 3, and 10 µM. For each concentrationof GDP, MgCl2 was run at 1, 3, 10, and 20 mM final concentration. The basic bufferwas 50 mM HEPES. Eu-GTP was used at a final concentration of 4 nM. The membraneamount used was 5 µg membrane protein/well. Incubation was performed for 1 h atroom temperature while mixing. Wells were filtered prior to washing 2X with WashBuffer and subsequent reading of the 615 nm emission on a Victor2V multilabel platereader.

PerkinElmer Life and Analytical Sciences

Human β2 adrenergic receptor (5 µg membrane protein) was incubated in buffercontaining: 50 mM HEPES, 0.1 µM GDP, 3 mM MgCl2, 4 nM Eu-GTP, and variousamounts of NaCl ranging from 0-200 mM in the presence or absence of 100 µMepinephrine for 60 min. Wells were filtered prior to washing 2X with Wash Buffer andsubsequent reading of the 615 nm emission on a Victor2V multilabel plate reader.

Results (cont.) 6

5

Saponin was added to final concentrations of 0, 1.2, 4, 12, 40, 120, and 400 µg/mL.In addition, the buffer contained 50 mM HEPES, 0.1 µM GDP, 3 mM MgCl2, and4 nM Eu-GTP. The membrane amount used was 5 µg/well. Wells were filtered priorto washing 2X with Wash Buffer and subsequent reading of the 615 nm emission ona Victor2V multilabel plate reader.

Titration of NaCl

Titration of Saponin

6PerkinElmer Life and Analytical Sciences

Results (cont.) 7

Various amounts of human β2 adrenergic receptor (0-20 µg membrane protein) wasincubated in buffer containing: 50 mM HEPES, 0.1 µM GDP, 3 mM MgCl2, 15 mMNaCl, 40 µg/mL saponin, and 4 nM Eu-GTP in the presence or absence of 100 µMepinephrine for 60 min. Wells were filtered prior to washing 2X with Wash Buffer andreading the 615 nm emission on a Victor2V multilabel plate reader.

Human β2 adrenergic receptor (5 µg membrane protein/well) was incubated in buffercontaining: 50 mM HEPES, 0.1 µM GDP, 3 mM MgCl2, 15 mM NaCl, 40 µg/mLsaponin, and 4 nM Eu-GTP in the presence or absence of 100 µM epinephrine fortimes ranging from 15 to 120 min. Wells were filtered prior to washing 2X with WashBuffer and reading the 615 nm emission on a Victor2V multilabel plate reader.

Titration of Membrane Amount

Determination of Optimal Incubation Time

PerkinElmer Life and Analytical Sciences

7

Conclusions

Easy to optimize, non-radioactive GTP binding assay

Previously shown to work with Gi-coupled receptors, now shown here to work with β2 adrenergic receptor, a Gs-coupled GPCR

Optimized conditions for Eu-GTP binding described

S/B = 2 obtained with as short as 60 min incubation

Assay can automated in combination with liquid handling to yield daily throughtput of 200 plates

8

www.perkinelmer.com

PerkinElmer Life and Analytical Sciences, 710 Bridgeport Avenue, Shelton, CT 06484 USA (800) 762-4000, (+1) (203) 925-4600

PerkinElmer Life and Analytical Sciences, Imperiastraat 8, BE-1930 Zaventem Belgium

Technical Support: in Europe: techsupport.europe@perkinelmer.com in US and Rest of World: techsupport@perkinelmer.com

Belgium: Tel: 0800 94 540 • France: Tel: 0800 90 77 62 • Netherlands: Tel: 0800 02 23 042 • Germany: Tel: 0800 1 81 00 32 • United Kingdom: Tel: 0800 89 60 46

Switzerland: Tel: 0800 55 50 27 • Italy: Tel: 800 79 03 10 • Sweden: Tel: 020 79 07 35 • Norway: Tel: 800 11 947 • Denmark: Tel: 80 88 3477 • Spain: Tel: 900 973 255

All trademarks or registered trademarks are the property of PerkinElmer, Inc.006775 (4465) 2003 PerkinElmer, Inc.