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Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs

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G Protein-Coupled Receptors in Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1335))

Abstract

We describe multiple bioorthogonal approaches to label G protein-coupled receptors (GPCRs) heterologously expressed in mammalian cells. The use of genetically encoded unnatural amino acids as bioorthogonal tags results in receptors that are expressed at lower levels than even their low abundance wild-type counterparts. Therefore, reproducible and sensitive quantification of the labeled GPCRs is extremely important and conventional methods are simply not sufficiently accurate and precise. Silver stains lack reproducibility, spectroscopic methods using fluorescent ligands are limited to quantifying only functional receptor molecules, and immunoassays using epitope tags derived from rhodopsin are particularly variable for low-abundance GPCRs. To avoid these shortcomings, we employ near infrared (NIR) imaging-based methods that enable simultaneous multi-color detection of two different antigens, thus facilitating the ratiometric analysis of bioorthogonally modified GPCRs. We anticipate that these multi-color detection strategies will provide new tools for quantitatively assessing stoichiometrically labeled GPCRs for studies of signalosomes and for structure–function relationships at a single molecule level.

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Acknowledgement

This work was supported by the Danica Foundation, the Crowley Family Fund, and an International Research Alliance at the Novo Nordisk Foundation Center for Basic Metabolic Research through an unconditional grant from the Novo Nordisk Foundation to the University of Copenhagen. H. T. was funded by the Tri-Institutional Program in Chemical Biology. We thank the Rockefeller University Proteomics Resource Center for peptide synthesis.

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Authors and Affiliations

  1. Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University, New York, NY, 10065, USA

    Minyoung Park, He Tian, Saranga Naganathan, Thomas P. Sakmar & Thomas Huber

  2. Department of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, 141 57, Huddinge, Sweden

    Thomas P. Sakmar

Authors
  1. Minyoung Park
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  2. He Tian
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  3. Saranga Naganathan
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  4. Thomas P. Sakmar
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  5. Thomas Huber
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Corresponding author

Correspondence to Thomas Huber .

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Editors and Affiliations

  1. Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Marta Filizola

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Park, M., Tian, H., Naganathan, S., Sakmar, T.P., Huber, T. (2015). Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs. In: Filizola, M. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 1335. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2914-6_6

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  • DOI: https://doi.org/10.1007/978-1-4939-2914-6_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2913-9

  • Online ISBN: 978-1-4939-2914-6

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