title: Multiplex detection of functional G protein-coupled receptors harboring site-specifically modified unnatural amino acids.
abstract: We developed a strategy for identifying positions in G protein-coupled receptors that are amenable to bioorthogonal modification with a peptide epitope tag under cell culturing conditions. We introduced the unnatural amino acid p-azido-l-phenylalanine (azF) into human CC chemokine receptor 5 (CCR5) at site-specific amber codon mutations. We then used strain-promoted azide-alkyne [3+2] cycloaddition to label the azF-CCR5 variants with a FLAG peptide epitope-conjugated aza-dibenzocyclooctyne (DBCO) reagent. A microtiter plate-based sandwich fluorophore-linked immunosorbent assay was used to probe simultaneously the FLAG epitope and the receptor using infrared dye-conjugated antibodies so that the extent of DBCO incorporation, corresponding nominally to labeling efficiency, could be quantified ratiometrically. The extent of incorporation of DBCO at the various sites was evaluated in the context of a recent crystal structure of maraviroc-bound CCR5. We observed that labeling efficiency varied dramatically depending on the topological location of the azF in CCR5. Interestingly, position 109 in transmembrane helix 3, located in a hydrophobic cavity on the extracellular side of the receptor, was labeled most efficiently. Because the bioorthogonal labeling and detection strategy described might be used to introduce a variety of different peptide epitopes or fluorophores into engineered expressed receptors, it might prove to be useful for a wide range of applications, including single-molecule detection studies of receptor trafficking and signaling mechanism.
authors.affiliation: Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University , New York, New York 10065, United States.
authors.forename: Saranga
authors.initials: S
authors.lastname: Naganathan
authors.forename: Sarmistha
authors.initials: S
authors.lastname: Ray-Saha
authors.forename: Minyoung
authors.initials: M
authors.lastname: Park
authors.forename: He
authors.initials: H
authors.lastname: Tian
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D003510:Cyclohexanes / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D000077592:Maraviroc; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D019713:Receptors, CCR5 / Q000737:chemistry / Q000378:metabolism*; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001386:Azides; D003510:Cyclohexanes; D000939:Epitopes; D005456:Fluorescent Dyes; D019713:Receptors, CCR5; D014230:Triazoles; C027162:4-azidophenylalanine; D010649:Phenylalanine; D000077592:Maraviroc