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Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

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Abstract

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.

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Fig. 1

Abbreviations

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CHS:

Cholesteryl hemisuccinate

DCM:

Dichloromethane

DDM:

Dodecyl-β-d-maltoside

EDTA:

Ethylenediaminetetraacetate

GPCR:

GTP-binding protein-coupled receptor

HPLC:

High pressure liquid chromatography

MBP:

Escherichia coli maltose binding protein

NT:

Neurotensin

NTR/NTS:

Neurotensin receptor

NTS-1A:

MBP-rT43NTR-TrxA-H10

SPR:

Surface plasmon resonance

TES:

Triethylsilane

TFA:

Trifluoroacetic acid

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Acknowledgements

We acknowledge the Medical Research Council (MRC), Biotechnology and Biological Sciences Research Council (BBSRC) and the UK Bionanotechnology IRC for funding this work. We thank GlaxoSmithKine (GSK), specifically Claus Spitzfaden, Martin Hibbs, Emma Jones, and Mark Hessey for allowing access to large scale fermentation, purification and SPR facilities. We would like to thank Dr. Simon Davis (Nuffield Department of Medicine, Oxford) for the use of his SPR machine.

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Authors and Affiliations

  1. Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Peter J. Harding, Timothy C. Hadingham & Anthony Watts

  2. The Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    James M. McDonnell

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  1. Peter J. Harding
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  2. Timothy C. Hadingham
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  3. James M. McDonnell
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  4. Anthony Watts
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Correspondence to Anthony Watts.

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Harding, P.J., Hadingham, T.C., McDonnell, J.M. et al. Direct analysis of a GPCR-agonist interaction by surface plasmon resonance. Eur Biophys J 35, 709–712 (2006). https://doi.org/10.1007/s00249-006-0070-x

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  • DOI: https://doi.org/10.1007/s00249-006-0070-x

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