title: Complex chimeras to map ligand binding sites of GPCRs.
abstract: Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.
authors.affiliation: Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. katy.l.gearing@gsk.com
authors.forename: K L
authors.initials: KL
authors.lastname: Gearing
authors.forename: A
authors.initials: A
authors.lastname: Barnes
authors.forename: J
authors.initials: J
authors.lastname: Barnett
authors.forename: A
authors.initials: A
authors.lastname: Brown
authors.forename: D
authors.initials: D
authors.lastname: Cousens
authors.forename: S
authors.initials: S
authors.lastname: Dowell
authors.forename: A
authors.initials: A
authors.lastname: Green
authors.forename: K
authors.initials: K
authors.lastname: Patel
authors.forename: P
authors.initials: P
authors.lastname: Thomas
authors.forename: F
authors.initials: F
authors.lastname: Volpe
authors.forename: F
authors.initials: F
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D002118:Calcium