title: Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers.
abstract: G protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor-ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.
authors: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.
authors: Amy
authors: A
authors: Grunbeck
authors: Thomas
authors: T
authors: Huber
authors: Thomas P
authors: TP
authors: Sakmar
mesh_terms: D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D003432:Cross-Linking Reagents; D043562:Receptors, G-Protein-Coupled