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G Protein-Coupled Receptors in Drug Discovery

Volume 1335 of the series Methods in Molecular Biology pp 67-93

Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs

  • Minyoung ParkAffiliated withLaboratory of Chemical Biology & Signal Transduction, The Rockefeller University
  • , He TianAffiliated withLaboratory of Chemical Biology & Signal Transduction, The Rockefeller University
  • , Saranga NaganathanAffiliated withLaboratory of Chemical Biology & Signal Transduction, The Rockefeller University
  • , Thomas P. SakmarAffiliated withLaboratory of Chemical Biology & Signal Transduction, The Rockefeller UniversityDepartment of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet
  • , Thomas HuberAffiliated withLaboratory of Chemical Biology & Signal Transduction, The Rockefeller University Email author 

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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