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Methods Enzymol

Title:		"Assessment of constitutive activity of a G protein-coupled receptor, CPR2, in Cryptococcus neoformans by heterologous and homologous methods"
Author(s):		Xue C; Wang Y; Hsueh YP;
Address:		"Public Health Research Institute, University of Medicine and Dentistry of New Jersey, Newark, New Jersey, USA"
Journal Title:		Methods Enzymol
Year:		2010
Volume:		484
Issue:
Page Number:		397 - 412
DOI:		10.1016/B978-0-12-381298-8.00020-4
ISSN/ISBN:		1557-7988 (Electronic) 0076-6879 (Print) 0076-6879 (Linking)
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"
Keywords:		"Animals Biological Assay/*methods Cryptococcus neoformans/genetics/*metabolism Receptors, G-Protein-Coupled/genetics/*metabolism;"
Notes:		"MedlineXue, Chaoyang Wang, Yina Hsueh, Yen-Ping eng R21 AI070230/AI/NIAID NIH HHS/ R21AI070230/AI/NIAID NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2010/11/03 Methods Enzymol. 2010; 484:397-412. doi: 10.1016/B978-0-12-381298-8.00020-4"