Application of Genetically-Encoded Photoactivatable Crosslinkers to Map Ligand-Binding Sites on G Protein-Coupled Receptors
type: CreativeWork
description: G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce biological signals. Although GPCRs represent the largest class of targets for therapeutic agents, ligand-binding sites have been precisely defined for only a small percentage of the receptors in the human genome. A general cellbased photocrosslinking approach was developed to investigate the binding interfaces necessary for the formation of GPCR signaling complexes. Amber codon suppression was extended to facilitate the incorporation of photoactivatable unnatural amino acids, p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine, into engineered GPCRs expressed in mammalian cells in culture. Proof-of-concept studies were carried out in chemokine receptors C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5), which are known HIV-1 co-receptors required for HIV-1 cellular entry in CD4+ cells. A cyclic peptide CXCR4-specific inhibitor, T140, photocrosslinked primarily to a specific site on CXCR4 and the result was reconciled with existing structural biology data. A small molecule drug, maraviroc, photocrosslinked to multiple sites on CCR5 and the results were extended to develop a computer homology model of the CCR5-maraviroc complex. In summary, the application of a novel targeted cellbased photocrosslinking strategy provided detailed information about receptor-ligand complexes in two chemokine receptors. The general approach described here using genetically-encoded photoreactive molecules to study the binding interactions between GPCRs and ligands represents a significant advance in the application of photocrosslinking reagents to address problems in biochemistry and pharmacology.
abstract: G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce biological signals. Although GPCRs represent the largest class of targets for therapeutic agents, ligand-binding sites have been precisely defined for only a small percentage of the receptors in the human genome. A general cellbased photocrosslinking approach was developed to investigate the binding interfaces necessary for the formation of GPCR signaling complexes. Amber codon suppression was extended to facilitate the incorporation of photoactivatable unnatural amino acids, p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine, into engineered GPCRs expressed in mammalian cells in culture. Proof-of-concept studies were carried out in chemokine receptors C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5), which are known HIV-1 co-receptors required for HIV-1 cellular entry in CD4+ cells. A cyclic peptide CXCR4-specific inhibitor, T140, photocrosslinked primarily to a specific site on CXCR4 and the result was reconciled with existing structural biology data. A small molecule drug, maraviroc, photocrosslinked to multiple sites on CCR5 and the results were extended to develop a computer homology model of the CCR5-maraviroc complex. In summary, the application of a novel targeted cellbased photocrosslinking strategy provided detailed information about receptor-ligand complexes in two chemokine receptors. The general approach described here using genetically-encoded photoreactive molecules to study the binding interactions between GPCRs and ligands represents a significant advance in the application of photocrosslinking reagents to address problems in biochemistry and pharmacology.
author: Grunbeck, Amy
keywords: G protein-coupled receptors (GPCRs), photo-cross-linking, CCR5, CXCR4, ligand binding, unnatural amino acids, Life Sciences
date published: 2013-01-01T08:00:00Z
date modified: 2026-07-24T04:11:45Z
provider: Rockefeller
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Rockefeller