Research Article | Published:

Micropatterned immobilization of a G protein–coupled receptor and direct detection of G protein activation

Nature Biotechnology volume 17, pages 11051108 (1999) | Download Citation

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Abstract

G protein–coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose–response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.

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Acknowledgements

We thank Dr. A. Brecht, D. Stamou, and Dr. R. Hovius for advice and critical reading of the manuscript; A. Heusler for the synthesis of the alkane thiols; and Dr. E. Delamarche (IBM, Zürich, Switzerland) for offering microcontact printing stamps. This research was supported by the Board of the Swiss Federal Institutes of Technology (SPP MINAST, 7.06) to H.V., the Deutsche Forschungsgemeinschaft (SFB 449) to K.P.H. and O.P.E., and the Fonds der Chemischen Industrie to K.P.H.

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Affiliations

  1. Swiss Federal Institute of Technology, Institute of Physical Chemistry, Laboratory for Physical Chemistry of Polymers and Membranes , CH-1015 Lausanne, Switzerland.

    • Christoph Bieri
    • , Stephan Heyse
    •  & Horst Vogel
  2. Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt-Universität zu Berlin , Germany.

    • Oliver P. Ernst
    •  & Klaus Peter Hofmann

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Correspondence to Horst Vogel.

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DOI

https://doi.org/10.1038/15090

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