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Investigation of G Protein-Coupled Receptor Function and Regulation Using Antisense

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G Protein-Coupled Receptor Genetics

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Delineation of GPCR function and regulation has relied heavily on the available pharmacological tools to manipulate GPCR activity, inhibit GPCR interacting proteins, or modulate downstream signaling molecules. Where appropriate agents are unobtainable, alternative molecular methods have been developed to determine the specific roles that individual proteins play in GPCR regulation and signaling. One such method utilizes RNA interference (RNAi) to suppress the expression of endogenous target proteins. The discovery that double-stranded (ds)RNA was able to deplete cellular protein expression paved the way for the development of modern RNAi methods. Initial experiments utilized long strands of dsRNA, which were cleaved via the action of an RNase named Dicer, into shorter 21–23 nucleotide RNAs, termed small interfering (si)RNAs. These active RNAs are unwound and incorporated into the RNA-induced silencing complex, wherein the antisense targeting strand binds to the complementary region of the target protein mRNA, promoting its subsequent destruction. Modern RNAi techniques mimic this process introducing carefully designed highly specific dsRNA constructs to effectively silence target gene expression within cells, offering an attractive alternative to the expensive development and maintenance of knockout animals. The use of RNAi has revolutionized the study of GPCR function and regulation circumventing the concerns over “off-target” effects which surround the overexpression of wild-type or inactive versions of target proteins. However, most importantly RNAi enables the precise identification of the roles that endogenous proteins play in cellular processes without the requirement for knockout animals. This chapter will examine how RNAi techniques have shed light on not only the function of GPCRs but also how gene silencing has uncovered novel roles for proteins that regulate GPCR responsiveness and signaling.

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Acknowledgments

The authors declare that they have no conflict of interest financial or otherwise. They would also like to thank the British Heart Foundation for continued funding.

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  1. Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester Royal Infirmary, Leicester, UK

    Jonathon Willets & Craig Nash

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  1. Jonathon Willets
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  2. Craig Nash
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  1. Oklahoma State University, Tulsa, Oklahoma, USA

    Craig W. Stevens

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Willets, J., Nash, C. (2014). Investigation of G Protein-Coupled Receptor Function and Regulation Using Antisense. In: Stevens, C. (eds) G Protein-Coupled Receptor Genetics. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-779-2_6

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  • DOI: https://doi.org/10.1007/978-1-62703-779-2_6

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