Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs
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.
Key wordsG protein-coupled receptor Bioorthogonal labeling SpAAC LI-COR Near infrared-based detection Quantitative analysis
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.
- 1.Khoury E, Clement S, Laporte SA (2014) Allosteric and biased G protein-coupled receptor signaling regulation: potentials for new therapeutics. Front Endocrinol 5:68Google Scholar
- 2.Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM (2013) Molecular signatures of G protein-coupled receptors. Nature 494:185–194Google Scholar
- 5.Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A, Cocucci E, Zurn A, Lohse MJ (2013) Single-molecule analysis of fluorescently labeled G protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci U S A 110:743–748Google Scholar
- 9.Lohse MJ, Nuber S, Hoffmann C (2012) Fluorescence/bioluminescence resonance energy transfer techniques to study G protein-coupled receptors activation and signaling. Pharmacol Rev 64:299–336Google Scholar
- 14.Ye S, Zaitseva E, Caltabiano G, Schertler GFX, Sakmar TP, Deupi X, Vogel R (2010) Tracking G protein-coupled receptors activation using genetically encoded infrared probes. Nature 464:1386–1390Google Scholar
- 18.Tian H, Naganathan S, Kazmi MA, Schwartz TW, Sakmar TP, Huber T (2014) Bioorthogonal fluorescent labeling of functional G protein-coupled receptors. Chem Bio Chem 15:1820–1829Google Scholar
- 24.Ray-Saha S, Huber T, Sakmar TP (2014) Antibody epitopes on G protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers. Biochemistry 53:1302–1310Google Scholar
- 27.Sasse J, Gallagher SR (2009) Staining proteins in gels. Curr Protoc Mol Biol 10(16):10.16.11–10.16.27Google Scholar
- 29.Twyman R (2013) Strategies for protein quantitation, 2nd Ed., Principles of Proteomics. Garland Science, New YorkGoogle Scholar
- 34.Mathews ST, Plaisance EP, Kim T (2009) Imaging systems for westerns: chemiluminescence vs. infrared detection. Methods Mol. Biol 536:499–513Google Scholar