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Assessment of Constitutive Activity of a G Protein-Coupled Receptor, Cpr2, in Cryptococcus neoformans by Heterologous and Homologous Methods

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. No.484. Elsevier , Amsterdam, pp. 397-412. ISBN 9780123812988. https://resolver.caltech.edu/CaltechAUTHORS:20110317-110558716

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Abstract

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
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/B978-0-12-381298-8.00020-4 DOIUNSPECIFIED
http://www.sciencedirect.com/science/article/B7CV2-51BNVR5-W/2/808347fd6e7520346e33b7271848dcd6PublisherUNSPECIFIED
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.
Funders:
Funding AgencyGrant Number
NIHAI070230
University of Medicine and Dentistry of New Jersey (UMDNJ)UNSPECIFIED
Series Name:Methods in Enzymology
Issue or Number:484
Record Number:CaltechAUTHORS:20110317-110558716
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20110317-110558716
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.
ID Code:22956
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Mar 2011 19:12
Last Modified:03 Oct 2019 02:42

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