Protein Engineering vol. 16 no. 5 pp. 365-372, 2003
© 2003 Oxford University Press
Complex chimeras to map ligand binding sites of GPCRs
Departments of 1Gene Expression and Protein Biochemistry, 4Systems Research, 5Screening Sciences, 6Computational Analytical and Structural Sciences and 7Bioinformatics, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK 3Present address: University of the West of England, Bristol, UK 8Present address: Department of Pharmacology, University of Cambridge, Cambridge, UK
2 To whom correspondence should be addressed. e-mail: katy.l.gearing{at}gsk.com
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 P2Y1, P2Y2 and BLT1 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 P2Y2/BLT1 chimera confirmed that the ligand binding determinants of BLT1 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.
Received April 20, 2002; revised March 31, 2003; accepted April 8, 2003.
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