Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs

  • Minyoung Park
  • He Tian
  • Saranga Naganathan
  • Thomas P. Sakmar
  • Thomas Huber
Part of the Methods in Molecular Biology book series (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.

Key words

G protein-coupled receptor Bioorthogonal labeling SpAAC LI-COR Near infrared-based detection Quantitative analysis 

Notes

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.

References

  1. 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. 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
  3. 3.
    Manglik A, Kobilka B (2014) The role of protein dynamics in GPCR function: insights from the beta2AR and rhodopsin. Curr Opin Cell Biol 27:136–143PubMedCrossRefGoogle Scholar
  4. 4.
    Huber T, Sakmar TP (2011) Escaping the flatlands: new approaches for studying the dynamic assembly and activation of GPCR signaling complexes. Trends Pharmacol Sci 32:410–419PubMedCentralPubMedCrossRefGoogle Scholar
  5. 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
  6. 6.
    Correa IR Jr (2014) Live-cell reporters for fluorescence imaging. Curr Opin Chem Biol 20:36–45PubMedCrossRefGoogle Scholar
  7. 7.
    Snaar-Jagalska BE, Cambi A, Schmidt T, de Keijzer S (2013) Single-molecule imaging technique to study the dynamic regulation of GPCR function at the plasma membrane. Methods Enzymol 521:47–67PubMedCrossRefGoogle Scholar
  8. 8.
    Xu X, Brzostowski JA, Jin T (2009) Monitoring dynamic GPCR signaling events using fluorescence microscopy, FRET imaging, and single-molecule imaging. Methods Mol Biol 571:371–383PubMedCrossRefGoogle Scholar
  9. 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
  10. 10.
    Sletten EM, Bertozzi CR (2009) Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed 48:6974–6998CrossRefGoogle Scholar
  11. 11.
    Huber T, Sakmar TP (2014) Chemical biology methods for investigating G protein-coupled receptor signaling. Chem Biol 21:1224–1237PubMedCrossRefGoogle Scholar
  12. 12.
    Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444PubMedCrossRefGoogle Scholar
  13. 13.
    Ye S, Huber T, Vogel R, Sakmar TP (2009) FTIR analysis of GPCR activation using azido probes. Nat Chem Biol 5:397–399PubMedCentralPubMedCrossRefGoogle Scholar
  14. 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
  15. 15.
    Ye S, Kohrer C, Huber T, Kazmi M, Sachdev P, Yan ECY, Bhagat A, RajBhandary UL, Sakmar TP (2008) Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J Biol Chem 283:1525–1533PubMedCrossRefGoogle Scholar
  16. 16.
    Tian H, Sakmar TP, Huber T (2013) Site-specific labeling of genetically encoded azido groups for multicolor, single-molecule fluorescence imaging of GPCRs. Methods Cell Biol 117:267–303PubMedCrossRefGoogle Scholar
  17. 17.
    Huber T, Naganathan S, Tian H, Ye S, Sakmar TP (2013) Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling. Methods Enzymol 520:281–305PubMedCrossRefGoogle Scholar
  18. 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
  19. 19.
    Naganathan S, Ye S, Sakmar TP, Huber T (2013) Site-specific epitope tagging of G protein-coupled receptors by Bioorthogonal modification of a genetically encoded unnatural amino acid. Biochemistry 52:1028–1036PubMedCrossRefGoogle Scholar
  20. 20.
    Naganathan S, Ray-Saha S, Park M, Tian H, Sakmar TP, Huber T (2015) Multiplex detection of functional GPCRs harboring site-specifically modified unnatural amino acids. Biochemistry 54(3):776–786PubMedCrossRefGoogle Scholar
  21. 21.
    Grunbeck A, Huber T, Sachdev P, Sakmar TP (2011) Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded Photocrosslinkers. Biochemistry 50:3411–3413PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Grunbeck A, Huber T, Abrol R, Trzaskowski B, Goddard WA III, Sakmar TP (2012) Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor. ACS Chem Biol 7:967–972PubMedCrossRefGoogle Scholar
  23. 23.
    Valentin-Hansen L, Park M, Huber T, Grunbeck A, Naganathan S, Schwartz TW, Sakmar TP (2014) Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid. J Biol Chem 289:18045–18054PubMedCentralPubMedCrossRefGoogle Scholar
  24. 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
  25. 25.
    Ramil CP, Lin Q (2013) Bioorthogonal chemistry: strategies and recent developments. Chem Commun 49:11007–11022CrossRefGoogle Scholar
  26. 26.
    Steinberg TH (2009) Protein gel staining methods: an introduction and overview. Methods Enzymol 463:541–563PubMedCrossRefGoogle Scholar
  27. 27.
    Sasse J, Gallagher SR (2009) Staining proteins in gels. Curr Protoc Mol Biol 10(16):10.16.11–10.16.27Google Scholar
  28. 28.
    Chevallet M, Luche S, Rabilloud T (2006) Silver staining of proteins in polyacrylamide gels. Nat Protoc 1:1852–1858PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Twyman R (2013) Strategies for protein quantitation, 2nd Ed., Principles of Proteomics. Garland Science, New YorkGoogle Scholar
  30. 30.
    Westermeier R, Marouga R (2005) Protein detection methods in proteomics research. Biosci Rep 25:19–32PubMedCrossRefGoogle Scholar
  31. 31.
    Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Cerretti DP, Urdal DL, Conlon PJ (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. BioTechnol 6:1204–1210CrossRefGoogle Scholar
  32. 32.
    Oprian DD, Molday RS, Kaufman RJ, Khorana HG (1987) Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci U S A 84:8874–8878PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    MacKenzie D, Arendt A, Hargrave P, McDowell JH, Molday RS (1984) Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides. Biochemistry 23:6544–6549PubMedCrossRefGoogle Scholar
  34. 34.
    Mathews ST, Plaisance EP, Kim T (2009) Imaging systems for westerns: chemiluminescence vs. infrared detection. Methods Mol. Biol 536:499–513Google Scholar
  35. 35.
    Knepp AM, Grunbeck A, Banerjee S, Sakmar TP, Huber T (2011) Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry 50:502–511PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Weldon S, Ambroz K, Schutz-Geschwender A, Olive DM (2008) Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis. Anal Biochem 375:156–158PubMedCrossRefGoogle Scholar
  37. 37.
    Franke RR, Sakmar TP, Oprian DD, Khorana HG (1988) A single amino acid substitution in rhodopsin (lysine 248 → leucine) prevents activation of transducin. J Biol Chem 263:2119–2122PubMedGoogle Scholar
  38. 38.
  39. 39.
  40. 40.
    Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Minyoung Park
    • 1
  • He Tian
    • 1
  • Saranga Naganathan
    • 1
  • Thomas P. Sakmar
    • 1
    • 2
  • Thomas Huber
    • 1
  1. 1.Laboratory of Chemical Biology & Signal TransductionThe Rockefeller UniversityNew YorkUSA
  2. 2.Department of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Center for Alzheimer ResearchKarolinska InstitutetHuddingeSweden

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