title: Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs.
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.
authors.affiliation: Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University, New York, NY, 10065, USA.
authors.forename: Minyoung
authors.initials: M
authors.lastname: Park
authors.forename: He
authors.initials: H
authors.lastname: Tian
authors.forename: Saranga
authors.initials: S
authors.lastname: Naganathan
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
mesh_terms: D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D003116:Color; D061565:Cycloaddition Reaction; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D061848:Optical Imaging / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D000480:Alkynes; D001386:Azides; D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; C027162:4-azidophenylalanine; D010649:Phenylalanine