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Approaches to Study GPCR Regulation in Native Systems

  • Jonathon M. WilletsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

The ability to assess whether individual proteins are involved in the signalling or regulation of G ­protein-coupled receptor signalling is highly dependent on the pharmacological tools available. In the absence of appropriate pharmacological agents, alternative molecular approaches have been developed to alter either protein function or expression. This has included the use of mutants, for example catalytically inactive (kinase-dead) enzymes, which when overexpressed function as dominant negatives to inhibit endogenous enzyme function, and more latterly small (21–23 bp) interfering RNA dsRNA oligos, whose antisense strand is designed complementary to the target protein mRNA and which can be used to deplete target protein expression. Critically, the success of these approaches depends on the transfection efficiency, and the chosen experimental assay in the cell type studied. Therefore, three transfection techniques and their merits and drawbacks are described. In addition, one method of examining G protein-coupled receptor (GPCR) regulation, combining siRNA-mediated GRK depletion and imaging of fluorescent GPCR ­signalling reporter biosensors in difficult-to-transfect cells is briefly described.

Key words

GPCR GRK siRNA Receptor regulation Dominant negative Cell transfection 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Cancer Studies and Molecular MedicineUniversity of LeicesterLeicesterUK

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