Abstract
A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G proteincoupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein α subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist / inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.
Keywords: assay development, g protein-coupled receptor, g protein, enzyme complementation, green fluorescent protein
Current Pharmaceutical Design
Title: G Protein-Coupled Receptor Fusion Proteins in Drug Discovery
Volume: 10 Issue: 17
Author(s): G. Milligan, G- J. Feng, R. J. Ward, N. Sartania, D. Ramsay, A. J. McLean and J. J. Carrillo
Affiliation:
Keywords: assay development, g protein-coupled receptor, g protein, enzyme complementation, green fluorescent protein
Abstract: A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G proteincoupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein α subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist / inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.
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Cite this article as:
Milligan G., Feng J. G-, Ward J. R., Sartania N., Ramsay D., McLean J. A. and Carrillo J. J., G Protein-Coupled Receptor Fusion Proteins in Drug Discovery, Current Pharmaceutical Design 2004; 10 (17) . https://dx.doi.org/10.2174/1381612043384295
DOI https://dx.doi.org/10.2174/1381612043384295 |
Print ISSN 1381-6128 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4286 |
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