Genetically-encoded molecular probes to study G protein-coupled receptors.
To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University.
Saranga
S
Naganathan
Amy
A
Grunbeck
He
H
Tian
Thomas
T
Huber
Thomas P
TP
Sakmar
D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D055668:Photochemical Processes; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014316:Tritium / Q000737:chemistry
D001386:Azides; D003432:Cross-Linking Reagents; D000939:Epitopes; D005456:Fluorescent Dyes; D008024:Ligands; D015335:Molecular Probes; D043562:Receptors, G-Protein-Coupled; D014316:Tritium; C027162:4-azidophenylalanine; D010649:Phenylalanine