Xue, Chaoyang and Wang, Yina and Hsueh, Yen-Ping (2010) Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterologous and Homologous Methods. In: Constitutive activity in receptors and other proteins. Part A. Methods in Enzymology (484). Elsevier , Amsterdam, pp. 397-412. ISBN 9780123812988 http://resolver.caltech.edu/CaltechAUTHORS:20110317-110558716
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G protein-coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and are primary targets for drug development. A variety of detection systems have been reported to study ligand–GPCR interactions. Using Saccharomyces cerevisiae to express foreign proteins has long been appreciated for its low cost, simplicity, and conserved cellular pathways. The yeast pheromone-responsive pathway has been utilized to assess a range of different GPCRs. We have identified a pheromone-like receptor, Cpr2, that is located outside of the MAT locus in the human fungal pathogen Cryptococcus neoformans. To characterize its function and potential ligands, we expressed CPR2 in a yeast heterologous expression system. To optimize for CPR2 expression in this system, pheromone receptor Ste3, regulator of G protein signaling (RGS) Sst2, and the cyclin-dependent kinase inhibitor Far1 were mutated. The lacZ gene was fused with the promoter of the FUS1 gene that is activated by the yeast pheromone signal and then introduced into yeast cells. Expression of CPR2 in this yeast heterologous expression system revealed that Cpr2 could activate the pheromone-responsive pathway without addition of potential ligands, suggesting it is a naturally occurring, constitutively active receptor. Mutation of a single amino acid, Leu^(222), was sufficient to reverse the constitutive activity of Cpr2. In this chapter, we summarize methods used for assessing the constitutive activity of Cpr2 and its mutants, which could be beneficial for other GPCR studies.
|Item Type:||Book Section|
|Additional Information:||© 2010 Elsevier Inc. Available online 29 October 2010. We thank Joe Heitman, Carol Newlon, and Tom Fowler for critical reading and comments on the chapter and Tom Fowler and James Konopka for S. cerevisiae strains and vectors. The original research on Cpr2 functional study was supported by National Institute of Health R21 grant (AI070230) to Joe Heitman. This work was supported by the new PI institutional start-up fund from UMDNJ to C. X.|
|Official Citation:||Chaoyang Xue, Yina Wang, Yen-Ping Hsueh, Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterologous and Homologous Methods, In: P. Michael Conn, Editor(s), Methods in Enzymology, Academic Press, 2010, Volume 484, Constitutive Activity in Receptors and Other Proteins, Part A, Pages 397-412, ISSN 0076-6879, ISBN 9780123812988, DOI: 10.1016/B978-0-12-381298-8.00020-4. (http://www.sciencedirect.com/science/article/B7CV2-51BNVR5-W/2/808347fd6e7520346e33b7271848dcd6)|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||17 Mar 2011 19:12|
|Last Modified:||17 Mar 2011 19:12|
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